Recently, Sugihara proposed an innovative causality concept, which, in contrast to statistical predictability in Granger sense, characterizes underlying deterministic causation of the system. This work exploits Sugihara causality analysis to develop novel EEG biomarkers for discriminating normal aging from mild cognitive impairment (MCI) and early Alzheimer's disease (AD). The hypothesis of this work is that scalp EEG based causality measurements have different distributions for different cognitive groups and hence the causality measurements can be used to distinguish between NC, MCI, and AD participants. The current results are based on 30-channel resting EEG records from 48 age-matched participants (mean age 75.7 years) — 15 normal controls (NCs), 16 MCI, and 17 early-stage AD. First, a reconstruction model is developed for each EEG channel, which predicts the signal in the current channel using data of the other 29 channels. The reconstruction model of the target channel is trained using NC, MCI, or AD records to generate an NC-, MCI-, or AD-specific model, respectively. To avoid over fitting, the training is based on the leave-one-out principle. Sugihara causality between the channels is described by a quality score based on comparison between the reconstructed signal and the original signal. The quality scores are studied for their potential as biomarkers to distinguish between the different cognitive groups. First, the dimension of the quality scores is reduced to two principal components. Then, a three-way classification based on the principal components is conducted. Accuracies of 95.8%, 95.8%, and 97.9% are achieved for resting eyes open, counting eyes closed, and resting eyes closed protocols, respectively. This work presents a novel application of Sugihara causality analysis to capture characteristic changes in EEG activity due to cognitive deficits. The developed method has excellent potential as individualized biomarkers in the detection of pathophysiological changes in early-stage AD.
•We explore EEG-based biomarkers for early Alzheimer's disease.•We investigate causality connectivity from scalp EEG in Sugihara sense.•Excellent diagnosis accuracies are achieved under three different protocol conditions.•We present the first biomedical application of Sugihara causality analysis.
Early Alzheimer's disease; Mild cognitive impairment; EEG-based diagnosis; Causality analysis
Hippocampal sclerosis of ageing is a prevalent brain disease that afflicts older persons and has been linked with cerebrovascular pathology. Arteriolosclerosis is a subtype of cerebrovascular pathology characterized by concentrically thickened arterioles. Here we report data from multiple large autopsy series (University of Kentucky Alzheimer’s Disease Centre, Nun Study, and National Alzheimer’s Coordinating Centre) showing a specific association between hippocampal sclerosis of ageing pathology and arteriolosclerosis. The present analyses incorporate 226 cases of autopsy-proven hippocampal sclerosis of ageing and 1792 controls. Case–control comparisons were performed including digital pathological assessments for detailed analyses of blood vessel morphology. We found no evidence of associations between hippocampal sclerosis of ageing pathology and lacunar infarcts, large infarcts, Circle of Willis atherosclerosis, or cerebral amyloid angiopathy. Individuals with hippocampal sclerosis of ageing pathology did not show increased rates of clinically documented hypertension, diabetes, or other cardiac risk factors. The correlation between arteriolosclerosis and hippocampal sclerosis of ageing pathology was strong in multiple brain regions outside of the hippocampus. For example, the presence of arteriolosclerosis in the frontal cortex (Brodmann area 9) was strongly associated with hippocampal sclerosis of ageing pathology (P < 0.001). This enables informative evaluation of anatomical regions outside of the hippocampus. To assess the morphology of brain microvasculature far more rigorously than what is possible using semi-quantitative pathological scoring, we applied digital pathological (Aperio ScanScope) methods on a subsample of frontal cortex sections from hippocampal sclerosis of ageing (n = 15) and control (n = 42) cases. Following technical studies to optimize immunostaining methods for small blood vessel visualization, our analyses focused on sections immunostained for smooth muscle actin (a marker of arterioles) and CD34 (an endothelial marker), with separate analyses on grey and white matter. A total of 43 834 smooth muscle actin-positive vascular profiles and 603 798 CD34-positive vascular profiles were evaluated. In frontal cortex of cases with hippocampal sclerosis of ageing, smooth muscle actin-immunoreactive arterioles had thicker walls (P < 0.05), larger perimeters (P < 0.03), and larger vessel areas (P < 0.03) than controls. Unlike the arterioles, CD34-immunoreactive capillaries had dimensions that were unchanged in cases with hippocampal sclerosis of ageing versus controls. Arteriolosclerosis appears specific to hippocampal sclerosis of ageing brains, because brains with Alzheimer’s disease pathology did not show the same morphological alterations. We conclude that there may be a pathogenetic change in aged human brain arterioles that impacts multiple brain areas and contributes to hippocampal sclerosis of ageing.
TDP-43; NACC; FTLD; SMA; HS-Ageing
Sphingosine kinase (SphK) is overexpressed by a variety of cancers, and its phosphorylation of sphingosine results in accumulation of sphingosine-1-phosphate (S1P) and activation of anti-apoptotic signal transduction. Existing data indicate a role for S1P in viral pathogenesis, but roles for SphK and S1P in virus-associated cancer progression have not been defined. Rare pathologic variants of diffuse large B-cell lymphoma arise preferentially in the setting of HIV infection, including primary effusion lymphoma (PEL), a highly mortal tumor etiologically linked to the Kaposi’s sarcoma-associated herpesvirus (KSHV). We have found that ABC294640, a novel clinical-grade small molecule selectively targeting SphK (SphK2 >> SphK1), induces dose-dependent caspase cleavage and apoptosis for KSHV+ patient-derived PEL cells, in part though inhibition of constitutive signal transduction associated with PEL cell proliferation and survival. These results were validated with induction of PEL cell apoptosis using SphK2-specific siRNA, as well as confirmation of drug-induced SphK inhibition in PEL cells with dose-dependent accumulation of pro-apoptotic ceramides and reduction of intracellular S1P. Furthermore, we demonstrate that systemic administration of ABC294640 induces tumor regression in an established human PEL xenograft model. Complimentary ex vivo analyses revealed suppression of signal transduction and increased KSHV lytic gene expression within drug-treated tumors, with the latter validated in vitro through demonstration of dose-dependent viral lytic gene expression within PEL cells exposed to ABC294640. Collectively, these results implicate interrelated mechanisms and SphK2 inhibition in the induction of PEL cell death by ABC294640 and rationalize evaluation of ABC294640 in clinical trials for the treatment of KSHV-associated lymphoma.
KSHV; sphingosine kinase; S1P; lymphoma; HIV
To evaluate the relationship between self-reported head injury and cognitive impairment, dementia, mortality, and Alzheimer’s (AD)-type pathological changes.
Clinical and neuropathological data from participants enrolled in a longitudinal study of aging and cognition (N=649) were analyzed to assess the chronic effects of self-reported head injury.
The effect of self-reported head injury on clinical state depends on age at assessment: for a 1-year increase in age, the
OR^ for transition to clinical MCI at the next visit for participants with a history of head injury is 1.21 and 1.34 for transition from MCI to dementia. Without respect to age, head injury increases the odds of mortality (
OR^=1.54). Head injury increases the odds of a pathological diagnosis of AD for men (
OR^=1.47) but not women (
OR^=1.18). Men with head injury have higher mean amyloid plaque counts in the neocortex and entorhinal cortex than men without.
Conclusions: Self-reported head injury is associated with earlier onset, increased risk of cognitive impairment and dementia, increased risk of mortality, and AD-type pathological changes.
head injury; Alzheimer’s disease; neuropathology; dementia; cognition
Depression is common in Parkinson’s disease and is associated with cognitive impairment. Dopaminergic medications are effective in treating the motor symptoms of Parkinson’s disease; however, little is known regarding the effects of dopaminergic pharmacotherapy on cognitive function in depressed Parkinson patients. This study examines the neuropsychological effects of dopaminergic pharmacotherapy in Parkinsonian depression. We compared cognitive function in depressed and non-depressed Parkinson patients at two time-points: following overnight withdrawal and after the usual morning regimen of dopaminergic medications. A total of 28 non-demented, right-handed patients with mild to moderate idiopathic Parkinson’s disease participated. Ten of these patients were depressed according to DSM IV criteria. Results revealed a statistically significant interaction between depression and medication status on three measures of verbal memory and a facial affect naming task. In all cases, depressed Parkinson’s patients performed significantly more poorly while on dopaminergic medication than while off. The opposite pattern emerged for the non-depressed Parkinson’s group. The administration of dopaminergic medication to depressed Parkinson patients may carry unintended risks.
Parkinson; mood; cognition; emotion; dopamine
Study of repeated learning mechanisms has been limited in amnestic mild cognitive impairment, a preclinical stage of Alzheimer disease modifiable by cognitive rehabilitation. We assessed repeated contextual working memory decline as an indicator of amnestic mild cognitive impairment in a sample of 45 older adults recruited from the tertiary care setting. Results indicated that contextual working memory impairment distinguished adults with preclinical disease from those without impairment despite similar overall cognitive performance, and comparison of the indicator with standard-of-care neuropsychological measures indicated discriminant validity. Contextual working memory impairment may represent a novel predictor of Alzheimer disease conversion risk.
Alzheimer disease; mild cognitive impairment; working memory; repetition priming; neuropsychological tests; cognitive therapy; aging
Sphingolipids are components of the plasma membrane whose metabolic manipulation is of interest as a potential therapeutic approach in a number of diseases. Sphingosine kinase 1 (SphK1), the major kinase that phosporylates sphingosine to sphingosine-1-phosphate (S1P), was previously shown by our group and others to modulate inflammation in murine models of inflammatory arthritis, inflammatory bowel disease and asthma. Sphingosine kinase 2’s (SphK2) impact on inflammation is less well known, as variable results were reported depending on the disease model. A specific SphK2 inhibitor inhibited inflammatory arthritis in one model, while siRNA knockdown of SphK2 worsened arthritis in another. We previously demonstrated that SphK1 deficient mice are protected against development of hTNF-α induced arthritis. To investigate the role of SphK2 in TNF-α induced arthritis, we developed SphK2 deficient hTNF-α overexpressing mice and separately treated hTNF-α mice with ABC294640, a SphK2 specific inhibitor. Our data show that genetic inhibition of SphK2 did not significantly impact the severity or progression of inflammatory arthritis, while pharmacologic inhibition of SphK2 led to significantly more severe arthritis. Compared to vehicle-treated mice, ABC294640 treated mice also had less S1P in whole blood and inflamed joint tissue, although the differences were not significant. ABC294640 treatment did not affect SphK1 activity in the inflamed joint while little SphK2 activity was detected in the joint. We conclude that the differences in the inflammatory phenotype in genetic inhibition vs. pharmacologic inhibition of SphK2 can be attributed to the amount of ABC294640 used in the experiments versus the impact of acute inhibition of SphK2 with ABC294640 vs. genetically-induced life-long SphK2 deficiency. Thus, inhibition of SphK2 appears to be proinflammatory in contrast to the clear anti-inflammatory effects of blocking SphK1. Therapies directed at this sphingosine kinase pathways will need to be specific in their targeting of sphingosine kinases.
Sphigosine kinase 2; inflammatory arthritis; sphingolipids; TNF
Sphingosine kinases (SKs) are promising new therapeutic targets for cancer because they regulate the balance between pro-apoptotic ceramides and mitogenic sphingosine-1-phosphate. The functions of the two SK isoenzymes, SK1 and SK2, are not redundant, with genetic ablation of SK2 having more pronounced anticancer effects than removal of SK1. Although several small molecule inhibitors of SKs have been described in the literature, detailed characterization of their molecular and cellular pharmacology, particularly their activities against human SK1 and SK2, have not been completed. Computational modeling of the putative active sites of SK1 and SK2 suggests structural differences that might allow isozyme-selective inhibitors. Therefore, we characterized several SK-inhibitory compounds which revealed differential inhibitory effects on SK1 and SK2 as follows: SKI-II and ABC294735 are SK1/2-dual inhibitors; CB5468139 is a SK1-selective inhibitor; and ABC294640 is a SK2-selective inhibitor. We examined the effects of the SK inhibitors on several biochemical and phenotypic processes in A498 kidney adenocarcinoma cells. The SK2-selective inhibitor ABC294640 demonstrated the most pronounced effects on SK1 and SK2 mRNA expression, decrease of S1P levels, elevation of ceramide levels, cell cycle arrest, and inhibition of proliferation, migration and invasion. ABC294640 also down-regulated the expression or activation of several signaling proteins, including STAT3, AKT, ERK, p21, p53 and FAK. These effects were equivalent or superior to responses to the SK1/2-dual inhibitors. Overall, these results suggest that inhibition of SK2 results in stronger anticancer effects than does inhibition of SK1 or both SK1 and SK2.
Hippocampal sclerosis of aging (HS-Aging) is a causative factor in a large proportion of elderly dementia cases. The current definition of HS-Aging rests on pathologic criteria: neuronal loss and gliosis in the hippocampal formation that is out of proportion to AD-type pathology. HS-Aging is also strongly associated with TDP-43 pathology. HS-Aging pathology appears to be most prevalent in the oldest-old: autopsy series indicate that 5–30 % of nonagenarians have HS-Aging pathology. Among prior studies, differences in study design have contributed to the study-to-study variability in reported disease prevalence. The presence of HS-Aging pathology correlates with significant cognitive impairment which is often misdiagnosed as AD clinically. The antemortem diagnosis is further confounded by other diseases linked to hippocampal atrophy including frontotemporal lobar degeneration and cerebrovascular pathologies. Recent advances characterizing the neurocognitive profile of HS-Aging patients have begun to provide clues that may help identify living individuals with HS-Aging pathology. Structural brain imaging studies of research subjects followed to autopsy reveal hippocampal atrophy that is substantially greater in people with eventual HS-Aging pathology, compared to those with AD pathology alone. Data are presented from individuals who were followed with neurocognitive and neuroradiologic measurements, followed by neuropathologic evaluation at the University of Kentucky. Finally, we discuss factors that are hypothesized to cause or modify the disease. We conclude that the published literature on HS-Aging provides strong evidence of an important and under-appreciated brain disease of aging. Unfortunately, there is no therapy or preventive strategy currently available.
TDP43; TDP-43; TARDBP; Dementia; Aging; Neuropathology; FTLD; Epidemiology; Genetics; Cognition; Neuroradiology; MRI; Hippocampus; Pathology; Arteriolosclerosis; Cerebrovascular; Oldest-old
Phosphorylation of sphingosine by sphingosine kinases (SphK1 and SphK2) generates sphingosine-1-phosphate (S1P), a bioactive sphingolipid which promotes cancer cell survival and tumor progression in vivo. We have recently reported that targeting SphK2 induces apoptosis for human primary effusion lymphoma (PEL) cell lines infected by the Kaposi’s sarcoma-associated herpesvirus (KSHV), and this occurs in part through inhibition of canonical NF-κB activation. In contrast, pharmacologic inhibition of SphK2 has minimal impact for uninfected B-cell lines or circulating human B cells from healthy donors. Therefore, we designed additional studies employing primary human endothelial cells to explore mechanisms responsible for the selective death observed for KSHV-infected cells during SphK2 targeting. Using RNA interference and a clinically relevant pharmacologic approach, we have found that targeting SphK2 induces apoptosis selectively for KSHV-infected endothelial cells through induction of viral lytic gene expression. Moreover, this effect occurs through repression of KSHV-microRNAs regulating viral latency and signal transduction, including miR-K12-1 which targets IκBα to facilitate activation of NF-κB, and ectopic expression of miR-K12-1 restores NF-κB activation and viability for KSHV-infected endothelial cells during SphK2 inhibition. These data illuminate a novel survival mechanism and potential therapeutic target for KSHV-infected endothelial cells: SphK2-associated maintenance of viral latency.
Research discoveries may lead to products for commercial development. A central consideration for the researcher is how involved s/he will be in the commercialization process. In some cases a university out-licenses the intellectual property, while in other cases the investigator may want to be involved in the development process and choose to start his or her own company to develop, and possibly to manufacture and sell the product. Before undertaking such a challenge, however, the investigator-turned-entrepreneur must consider a variety of issues, including: career goals, financial and time commitments, potential conflicts of interest and/or commitment, start-up funding, as well as his or her ability to run a company or step aside to allow business experts to make necessary decisions. This article discusses some personal considerations in deciding to start a spin-out company and provides information on some of the available government grants to assist you should you decide to undertake your product’s commercial development. In particular, the Small Business Innovative Research and Small Business Technology Transfer programs of federal funding agencies are often the source of very early funding for new biomedical companies.
Intellectual property; technology commercialization; faculty spin-out company; SBIR/STTR
Volume losses in the medial temporal lobe, posterior cingulated, and orbitofrontal region have been observed in Alzheimer’s disease (AD). Smaller reductions in similar regions have also been reported in amnestic mild cognitive impairment (aMCI), a canonical precursor to AD. We previously demonstrated that volume loss in bilateral anteromedial temporal lobe is present at baseline in longitudinally followed normal subjects who later developed MCI or AD. In this study we compared grey matter volumes within this predefined anteromedial temporal region (AMTR) at baseline between: 1) normal subjects enrolled in the Alzheimer’s Disease Neuroimaging Initiative (ADNI) who subsequently developed cognitive complaints as reflected in a CDR memory box score of 0.5; and 2) normal subjects who remained normal over a median of 48 months of follow-up (CDR sum of boxes 0). We found significantly decreased volume within AMTR in the ADNI memory complainers. To relate AMTR results to those from conventional anatomy, we demonstrate that volumes extracted with the ICBM amygdala region had the best correspondence with AMTR volumes. In contrast, regions that have demonstrated volume loss in frank MCI and AD in ADNI, e.g., the posterior cingulate, did not show volume loss. These findings provide independent confirmation that volume changes preceding MCI occur in AMTR, a region of overlap between amygdala and anterior hippocampus.
Alzheimer’s Disease Neuroimaging Initiative; Alzheimer’s disease risk; amygdala; brain aging; hippocampus; longitudinal studies; magnetic resonance imaging; medial temporal lobe; structural neuroimaging; voxel-based morphometry
Vascular dementia (VaD) is the second leading cause of dementia behind Alzheimer's disease (AD) and is a frequent comorbidity with AD, estimated to occur in as many as 40% of AD patients. The causes of VaD are varied and include chronic cerebral hypoperfusion, microhemorrhages, hemorrhagic infarcts, or ischemic infarcts. We have developed a model of VaD by inducing hyperhomocysteinemia (HHcy) in wild-type mice. By placing wild-type mice on a diet deficient in folate, B6, and B12 and supplemented with excess methionine, we induced a moderate HHcy (plasma level homocysteine 82.93±3.561 μmol). After 11 weeks on the diet, the hyperhomocysteinemic mice showed a spatial memory deficit as assessed by the 2-day radial-arm water maze. Also, magnetic resonance imaging and subsequent histology revealed significant microhemorrhage occurrence. We found neuroinflammation induced in the hyperhomocysteinemic mice as determined by elevated interleukin (IL)-1β, tumor necrosis factor (TNF)α, and IL-6 in brain tissue. Finally, we found increased expression and increased activity of the matrix metalloproteinase 2 (MMP2) and MMP9 systems that are heavily implicated in the pathogenesis of cerebral hemorrhage. Overall, we have developed a dietary model of VaD that will be valuable for studying the pathophysiology of VaD and also for studying the comorbidity of VaD with other dementias and other neurodegenerative disorders.
animal models; hemosiderin; homocysteinemia; inflammation; vascular dementia
VCP disease associated with Inclusion body myopathy, Paget disease of the bone and frontotemporal dementia is a progressive autosomal dominant disorder caused by mutations in Valosin containing protein gene. To establish genotype-phenotype correlations we analyzed clinical and biochemical markers from a database of 190 members in 27 families harboring ten missense mutations. Individuals were grouped into three categories: symptomatic, presymptomatic carriers and non-carriers. The symptomatic families were further divided into ten groups based on their VCP mutations. There was marked intra and inter-familial variation; and significant genotype-phenotype correlations were difficult because of small numbers. Nevertheless when comparing the two most common mutations, R155C mutation was found to be more severe, with earlier onset of myopathy and Paget (p=0.03).
Survival analysis of all subjects revealed an average life span after diagnosis of myopathy and Paget of 18 and 19 years respectively, and after dementia only 6 years. R155C had a reduced survival compared to the R155H mutation (p=0.03). We identified amyotrophic lateral sclerosis (ALS) in thirteen individuals (8.9%) and Parkinson’s disease in five individuals (3%); however there was no genotypic correlation. This study represents the largest dataset of patients with VCP disease and expands our understanding of natural history and provides genotype-phenotype correlations in this unique disease.
amyotrophic lateral sclerosis; frontotemporal dementia; genotype-phenotype; inclusion body myopathy; Paget’s disease of bone; valosin containing protein
In this review focus is on structural imaging in the Alzheimer’s disease pre-states, particularly cognitively normal (CN) persons at future dementia risk. Findings in mild cognitive impairment (MCI) are described here only for comparison with CN. Cited literature evidence and commentary address issues of structural imaging alterations in CN that precede MCI and AD, regional patterns of such alterations, and the time relationship between structural imaging alterations and the appearance of symptoms of AD, issues relevant to the conduct of future AD prevention trials.
review; human; mild cognitive impairment; Alzheimer’s disease; magnetic resonance imaging; morphometry; cognitive normality; normal aging
Compounds 1-4 were previously reported as potent antimitotic and antitumor agents with Pgp modulatory effects. Compounds 5-18 have been synthesized in an attempt to optimize the various activities of 1-4. Compounds 5-10 explored the influence of methoxy substitutions on the 7-benzyl moiety in 1, while 11-18 investigated the influence of incorporation of a sulfur linker at C5 compared to 1-3. Compounds 5-10 demonstrated potent single-digit micromolar tumor cell cytotoxicity, Pgp modulation and microtubule inhibition. Compound 7 of this series was the most potent and showed GI50 values in the nanomolar range against several human tumor cell lines in the standard NCI preclinical in vitro screen. Antitumor activity and Pgp modulatory effects were found to decrease for the 5-phenylthio compounds 11-14 compared to their 5-phenylethyl analogs 2-4 and the standard compound Taxol. Incorporation of methoxy substitutions on the 7-benzyl moiety improved antitumor activity for the 5-phenylthio compounds 16 and 17. Compounds 16 and 17 demonstrated single to two-digit micromolar inhibition of tumor cells.
Pyrrolo[2,3-d]pyrimidines; Microtubule inhibitors; Microwave assisted organic synthesis
The ceramide/sphingosine-1-phosphate (S1P) rheostat has been hypothesized to play a critical role in regulating tumor cell fate, with elevated levels of ceramide inducing death and elevated levels of S1P leading to survival and proliferation. Ceramidases are key enzymes that control this rheostat by hydrolyzing ceramide to produce sphingosine, and may also confer resistance to drugs and radiation. Therefore, ceramidase inhibitors have excellent potential for development as new anticancer drugs. In this study, we identify a novel ceramidase inhibitor (Ceranib-1) by screening a small molecule library and describe the synthesis of a more potent analog (Ceranib-2). In a cell-based assay, both compounds were found to inhibit cellular ceramidase activity toward an exogenous ceramide analog, induce the accumulation of multiple ceramide species, decrease levels of sphingosine and S1P, inhibit the proliferation of cells alone and in combination with paclitaxel, and induce cell cycle arrest and cell death. In vivo, Ceranib-2 was found to delay tumor growth in a syngeneic tumor model without hematologic suppression or overt signs of toxicity. These data support the selection of ceramidases as suitable targets for anticancer drug development, and provide the first non-lipid inhibitors of human ceramidase activity.
Ceramidase; Ceramide; Inhibitor; Sphingosine; Tumor
Sphingosine kinases (SK) regulate the balance between pro-apoptotic ceramides and mitogenic sphingosine-1-phosphate (S1P); however, the functions of the two isoenzymes (SK1 and SK2) in tumor cells are not well defined. Therefore, RNA interference was used to assess the individual roles of SK1 and SK2 in tumor cell sphingolipid metabolism, proliferation and migration/invasion. Treatment of A498, Caki-1 or MDA-MB-231 cells with siRNA specific for SK1 or SK2 effectively suppressed the expression of the target mRNA and protein. Ablation of SK1 did not affect mRNA or protein levels of SK2, and reduced intracellular levels of S1P while elevating ceramide levels. In contrast, ablation of SK2 elevated mRNA, protein and activity levels of SK1, and increased cellular S1P levels. Interestingly, cell proliferation and migration/invasion were suppressed more by SK2-selective ablation than by SK1-selective ablation, demonstrating that the increased S1P does not rescue these phenotypes. Similarly, exogenous S1P did not rescue the cells from the anti-proliferative or anti-migratory effects of the siRNAs. Consistent with these results, differential affects of SK1- and SK2-selective siRNAs on signaling proteins including p53, p21, ERK1, ERK2, FAK and VCAM1 indicate that SK1 and SK2 have only partially overlapping functions in tumor cells. Overall, these data indicate that loss of SK2 has stronger anticancer effects than does suppression of SK1. Consequently, selective inhibitors of SK2 may provide optimal targeting of this pathway in cancer chemotherapy.
Sphingosine Kinase; Isoenzyme; siRNA; Proliferation; Migration; Anticancer
We have discovered a novel series of 7-benzyl-4-methyl-5-[(2-substituted phenyl)ethyl]-7H-pyrrolo[2,3-d]-pyrimidin-2-amines, which possess antimitotic and antitumor activities against antimitotic-sensitive as well as resistant tumor cells. These agents bind to a site on tubulin that is distinct from the colchicine, vinca alkaloid, and paclitaxel binding sites and some, in addition to their antitumor activity, remarkably also reverse tumor resistance to antimitotic agents mediated via the P-glycoprotein efflux pump. The compounds were synthesized from N-(7-benzyl-5-ethynyl-4-methyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-2,2-dimethylpro-panamide 11 or the corresponding 5-iodo analog 14 via Sonogashira couplings with appropriate iodobenzenes or phenylacetylene followed by reduction and deprotection to afford the target analogs. Sodium and liquid NH3 afforded the debenzylated analogs. The most potent analog 1 was one to three digit nanomolar against the growth of both sensitive and resistant tumor cells in culture. Compounds of this series are promising novel antimitotic agents that have the potential for treating both sensitive and resistant tumors.
The balance between the pro-apoptotic lipids ceramide and sphingosine and the pro-survival lipid sphingosine 1-phosphate (S1P) is termed the “sphingosine rheostat”. Two isozymes, sphingosine kinase 1 and 2 (SK1 and SK2), are responsible for phosphorylation of pro-apoptotic sphingosine to form pro-survival S1P. We have previously reported the antitumor properties of an SK2 selective inhibitor, ABC294640, alone or in combination with the multikinase inhibitor sorafenib in mouse models of kidney carcinoma and pancreatic adenocarcinoma. Here, we evaluated the combined antitumor effects of the aforementioned drug combination in two mouse models of hepatocellular carcinoma. Although combining the SK2 inhibitor, ABC294640 and sorafenib in vitro only afforded additive drug-drug effects, their combined antitumor properties in the mouse model bearing HepG2 cells mirrored effects previously observed in animals bearing kidney carcinoma and pancreatic adenocarcinoma cells. Combining ABC294640 and sorafenib led to a decrease in the levels of phosphorylated ERK in SK-HEP -1 cells, indicating that the antitumor effect of this drug combination is likely mediated through a suppression of the MAPK pathway in hepatocellular models. We also measured levels of S1P in the plasma of mice treated with two different doses of ABC294640 and sorafenib. We found decreases in the levels of S1P in plasma of mice treated daily with 100 mg/kg of ABC294640 for 5 weeks, and this decrease was not affected by coadministration of sorafenib. Taken together, these data support combining ABC294640 and sorafenib in clinical trials in HCC patients. Furthermore, monitoring levels of S1P may provide a pharmacodynamic marker of ABC294640 activity.
pharmacodynamics; targeted therapy; sphingosine kinase; hepatocellular carcinoma
Approximately half of tumor cell lines are resistant to the tumor-selective apoptotic effects of tumor necrosis factor-related apoptosis-inducing ligand (Apo22L/TRAIL). Previously, we showed that combining Apo2L/TRAIL with sorafenib, a multikinase inhibitor, results in dramatic efficacy in Apo2L/TRAIL-resistant tumor xenografts via inhibition of Mcl-1. Soluble Apo2L/TRAIL is capable of binding to several surface receptors, including the pro-apoptotic death receptors, DR4 and DR5, and decoy receptors, DcR1 and DcR2. Monoclonal antibodies targeting either of these death receptors are being investigated as antitumor agents in clinical trials. We hypothesized that sorafenib and Apo2L/TRAIL or Apo2L/TRAIL death receptor agonist (TRA) antibodies against DR4 (mapatumumab) and DR5 (lexatumumab) will overcome resistance to Apo2L/TRAIL-mediated apoptosis and as increase antitumor efficacy in Apo2L/TRAIL-sensitive solid tumors.
We found that Apo2L/TRAIL or TRA antibodies combined with sorafenib synergistically reduce cell growth and increase cell death across a panel of solid tumor cell lines in vitro. This panel included human breast, prostate, colon, liver and thyroid cancers. The cooperativity of these combinations was also observed in
vivo, as measured by tumor volume and TUNEL staining as a measure of apoptosis. We found that sorafenib inhibits Jak/Stat3 signaling and downregulates their target genes, including cyclin D1, cyclin D2 and Mcl-1, in a dose-dependent manner.
The combination of sorafenib with Apo2L/TRAIL or Apo2L/TRAIL receptor agonist antibodies sensitizes Apo2L/TRAIL-resistant cells and increases the sensitivity of Apo2L/TRAIL-sensitive cells. Our findings demonstrate the involvement of the Jak2-Stat3-Mcl1 axis in response to sorafenib treatment, which may play a key role in sorafenib-mediated sensitization to Apo2L/TRAIL.
Pro-inflammatory cytokines and growth factors such as vascular endothelial growth factor (VEGF) contribute to the loss of the blood-retinal barrier (BRB) and subsequent macular edema in various retinal pathologies. VEGF signaling requires conventional PKC (PKCβ) activity; however, PKCβ inhibition only partially prevents VEGF-induced endothelial permeability and does not affect pro-inflammatory cytokine-induced permeability suggesting the involvement of alternative signaling pathways. Here, we provide evidence for the involvement of atypical protein kinase C (aPKC) signaling in VEGF-induced endothelial permeability and identify a novel class of inhibitors of aPKC that prevent BRB breakdown in vivo. Genetic and pharmacological manipulations of aPKC isoforms were used to assess their contribution to endothelial permeability in culture. A chemical library was screened using an in vitro kinase assay to identify novel small molecule inhibitors and further medicinal chemistry was performed to delineate a novel pharmacophore. We demonstrate that aPKC isoforms are both sufficient and required for VEGF-induced endothelial permeability. Furthermore, these specific, potent, non-competitive, small molecule inhibitors prevented VEGF-induced tight junction internalization and retinal endothelial permeability in response to VEGF in both primary culture and in rodent retina. These data suggest that aPKC inhibition with 2-amino-4-phenyl-thiophene derivatives may be developed to preserve the BRB in retinal diseases such as diabetic retinopathy or uveitis and the blood-brain barrier (BBB) in the presence of brain tumors.
vascular endothelial growth factor (VEGF); atypical protein kinase C (aPKC); blood-retinal barrier (BRB); blood-brain barrier (BBB)
Sphingolipid metabolism is driven by inflammatory cytokines. These cascade of events include the activation of sphingosine kinase (SK), and subsequent production of the mitogenic and proinflammatory lipid sphingosine 1-phosphate (S1P). Overall, S1P is one of the crucial components in inflammation, making SK an excellent target for the development of new anti-inflammatory drugs. We have recently shown that SK inhibitors suppress colitis and hypothesize here that the novel SK inhibitor, ABC294640, prevents the development of colon cancer. In an azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model, there was a dose-dependent decrease in tumor incidence with SK inhibitor treatment. The tumor incidence (number of animals with tumors per group) in the vehicle, ABC294640 (20 mg/kg) and ABC294640 (50 mg/kg) groups were 80, 40 and 30%, respectively. Tumor multiplicity (number of tumors per animal) also decreased from 2.1 ± 0.23 tumors per animal in the AOM + DSS + vehicle group to 1.2 ± 0 tumors per animal in the AOM + DSS + ABC294640 (20 mg/kg) and to 0.8 ± 0.4 tumors per animal in the AOM + DSS + ABC294640 (50 mg/kg) group. Importantly, with ABC294640, there were no observed toxic side effects. To explore mechanisms, we isolated cells from the colon (CD45−, representing primarily colon epithelial cells) and (CD45+, representing primarily colon inflammatory cells) then measured known targets of SK that control cell survival. Results are consistent with the hypothesis that the inhibition of SK activity by our novel SK inhibitor modulates key pathways involved in cell survival and may be a viable treatment strategy for the chemoprevention colitis-driven colon cancer.
Palmitoylation is required for the activities of several cancer-associated proteins, making the palmitoyl acyltransferase (PAT) enzymes that catalyze these reactions potential targets for anticancer therapeutics. In this study, we sought to identify and characterize a human PAT with activity toward N-terminally myristoylated and palmitoylated proteins. NIH/3t3 cells were stably transfected with vectors containing no insert, wild type human DHHC20, or a serine-substituted DHHS20 mutant. Compared with control cells, cells overexpressing wild-type DHHC20 displayed an increase in palmitoylation activity toward a peptide that mimics the N-terminus of myristoylated and palmitoylated proteins, but had no change in activity toward a peptide that mimics the C-terminus of farnesylated and palmitoylated proteins. Cells expressing DHHS20 had no significant change in activity toward either peptide. Overexpression of DHHC20 also caused phenotypic changes consistent with cellular transformation, including colony formation in soft agar, decreased contact inhibition of growth, and increased proliferation under low-serum conditions. Quantitative polymerase chain reaction analyses of human tissues demonstrated that DHHC20 is expressed in a tissue-specific manner, and is overexpressed in several types of human tumors, including ovarian, breast and prostate. Overall, these results demonstrate that DHHC20 is a human N-terminal-myristoyl-directed PAT involved in cellular transformation, that may play a role in cancer.
DHHC; myristoyl; palmitoyl acyltransferase; palmitoylation; proliferation
Recent behavioral data have shown that lifelong bilingualism can maintain youthful cognitive control abilities in aging. Here, we provide the first direct evidence of a neural basis for the bilingual cognitive control boost in aging. Two experiments were conducted, using a perceptual task switching paradigm, and including a total of 110 participants. In Experiment 1, older adult bilinguals showed better perceptual switching performance than their monolingual peers. In Experiment 2, younger and older adult monolinguals and bilinguals completed the same perceptual task switching experiment while fMRI was performed. Typical age-related performance reductions and fMRI activation increases were observed. However, like younger adults, bilingual older adults outperformed their monolingual peers while displaying decreased activation in left lateral frontal cortex and cingulate cortex. Critically, this attenuation of age-related over-recruitment associated with bilingualism was directly correlated with better task switching performance. In addition, the lower BOLD response in frontal regions accounted for 82% of the variance in the bilingual task switching reaction time advantage. These results suggest that lifelong bilingualism offsets age-related declines in the neural efficiency for cognitive control processes.