Changes In Cytoarchitecture Research Articles

P5.77 Sarcomeric gene and protein expression during human myogenesis in vitro

In vertebrates, skeletal muscle is found throughout the body and forms during the entire life span. Adult skeletal muscle displays a high regenerative capacity, with satellite cells as the primary source of this remarkable ability. Satellite cells are quiescent muscle progenitors located between myofibers and their surrounding basal lamina. The formation of skeletal muscle cells, myogenesis, is a complex process, which takes place during embryonic development, postnatal growth and adult muscle regeneration. Myogenesis is marked by specific changes in muscle cell morphology and cytoarchitecture through the fusion of mononucleated myoblasts into multinucleated myotubes and the assembly of contractile sarcomeric myofibrils. The formation of contractile myofibrils involves the transition of myoblast cytoarchitecture, which requires a change in the expression of proteins from non-muscle-specific to muscle-specific isoforms. In this study, combined molecular and immunocytochemistry methods were used to investigate the expression profile of sarcomeric genes and proteins during myoblast proliferation and differentiation in human muscle cell line. We observed the expression of a panel of sarcomeric gene transcripts together with the early expression of a number of the investigated sarcomeric proteins at the proliferated myoblasts. In addition, after 6 days of differentiation, the majority of cells expressed all the investigated sarcomeric proteins. The results presented here provide novel insights into the sequential onset of the sarcomeric genes and proteins and support the previous notion that non-committed stem cells exist in the adult muscle satellite cells during myogenesis in vitro. In addition, our study demonstrates the effectiveness of a differentiation system, which transforms undifferentiated and star-shaped myoblasts, into long multinucleated myotubes, displaying the features of contractile muscle cells.

The relationship between callosal axons and cortical neurons in the planum temporale: Alterations in schizophrenia

The relationship between “connectivity” measures such as DTI and the cellular alterations in the cortex that give rise to those connections remains unclear. Cytoarchitectural changes in the planum temporale (PT) suggest impaired layer III feedforward projection neurons in schizophrenia. Altered hemispheric asymmetry of the PT has been reported in patients, along with altered white matter density in the corpus callosum, and there is anomalous activation of the PT during auditory hallucinations. We measured layer III cell density and pyramidal neuron size in PT of both hemispheres of post-mortem brains from patients with schizophrenia ( n = 16) and control subjects ( n = 16). We found reduced cell density and the loss of a correlation between magnopyramidal neuron density and axon number in the isthmus of the corpus callosum in schizophrenia. The normal asymmetry indicated that magnopyramidal neurons tend towards being larger and denser in the left PT but this asymmetry is significantly reduced in schizophrenia. The findings offer cytoarchitectural insight into the relationship between PT cortex and callosal white matter abnormalities in schizophrenia.

TAT-mediated transduction of MafA protein in utero results in enhanced pancreatic insulin expression and changes in islet morphology.

Alongside Pdx1 and Beta2/NeuroD, the transcription factor MafA has been shown to be instrumental in the maintenance of the beta cell phenotype. Indeed, a combination of MafA, Pdx1 and Ngn3 (an upstream regulator of Beta2/NeuroD) was recently reported to lead to the effective reprogramming of acinar cells into insulin-producing beta cells. These experiments set the stage for the development of new strategies to address the impairment of glycemic control in diabetic patients. However, the clinical applicability of reprogramming in this context is deemed to be poor due to the need to use viral vehicles for the delivery of the above factors. Here we describe a recombinant transducible version of the MafA protein (TAT-MafA) that penetrates across cell membranes with an efficiency of 100% and binds to the insulin promoter in vitro. When injected in utero into living mouse embryos, TAT-MafA significantly up-regulates target genes and induces enhanced insulin production as well as cytoarchitectural changes consistent with faster islet maturation. As the latest addition to our armamentarium of transducible proteins (which already includes Pdx1 and Ngn3), the purification and characterization of a functional TAT-MafA protein opens the door to prospective therapeutic uses that circumvent the use of viral delivery. To our knowledge, this is also the first report on the use of protein transduction in utero.

Osmotic stabilizer-coupled suppression of NDR defects is dependent on the calcium–calcineurin signaling cascade in Aspergillus nidulans

Establishment and maintenance of cell polarity are coordinated by signaling pathways such as NDR (nuclear Dbf2-related) protein-kinase signaling and calcium signaling pathway. The NDR family of kinase is structurally related to the human myotonic dystrophy kinase, which, when impaired, confers a disease that involves changes in cytoarchitecture and ion homeostasis. CotA kinase, a member of the NDR protein kinase family, forms a complex with MobB to regulate cell polarized growth in Aspergillus nidulans. Our previous study demonstrated that mobB/ cotA defects could be suppressed by the osmotic stress in the presence of external calcium. In this study, via the genetic and molecular approach, we further demonstrated that Ca 2+-permeable stretch-activated nonselective cation channel-MidA, calcium/calmodulin-dependent protein phosphatase catalatic subunit-CnaA and external calcium, but not voltage-gated calcium channel homolog-CchA, were required for the osmotic stabilizer-coupled suppression. The up-regulation of calcium/calcineurin signaling pathway induced by osmotic stress might be the reason for bypassing the requirements of NDR kinase complex, which is otherwise necessary for polar morphogenesis. Our results suggest that calcium–calcineurin signaling pathway coordinates with MobB/CotA kinase complex in regulating polarity growth via maintaining cellular calcium homeostasis. However, CchA may act differently as other components in calcium signaling pathway in Aspergillus nidulans. These findings provide an excellent opportunity to identify the potential pathway linking NDR protein-kinase network to calcium signaling pathway.

Egocentric working memory impairment and dendritic spine plastic changes in prefrontal neurons after NMDA receptor blockade in rats

Working memory may involve context-dependent allocentric or own movement-dependent egocentric strategies. While allocentric working memory can be disrupted by N-methyl-D-aspartate (NMDA) blockage, the possible effects of NMDA receptor manipulation on the egocentric strategy have not been studied. Because dendritic spine plasticity in part underlies working memory-related behavioral efficiency, egocentric working memory performance was evaluated in adult rats following NMDA receptor blockade with 10 mg/kg of the NMDA-receptor antagonist CPP, i.p. Dendritic spine density and the proportion of different spine types (thin, stubby, mushroom, wide, branched and double) were assessed in third-layer pyramidal neurons of the dorsomedial prefrontal cortex, after behavioral testing. Working memory was evaluated by challenging the rats to resolve twelve trials per day in a single-day session over five consecutive days, in a “cross-arm” maze and according to a delayed match-to-sample procedure. In control animals, the dendritic spine density remained unchanged after behavioral testing, although the proportion of mushroom spines decreased while that of the branched spines increased. NMDA receptor blockade impaired the behavioral performance of rats and resulted in a decrease in dendritic spine density when compared to the control animals, and dendritic spine types were unchanged. These results suggest that behavioral efficiency of egocentric working memory is dependent on NMDA receptor activation, and that plastic changes in spine cytoarchitecture may play a key role in behavioral performance.

Age-related changes of neuron numbers in the frontal cortex of a transgenic mouse model of Alzheimer’s disease

Alzheimer’s disease (AD) is a neurodegenerative disorder, characterized by amyloid plaque accumulation, intracellular tangles and neuronal loss in selective brain regions. The frontal cortex, important for executive functioning, is one of the regions that are affected. Here, we investigated the neurodegenerative effects of mutant human amyloid precursor protein (APP) and presenilin 1 (PS1) on frontal cortex neurons in APP/PS1KI mice, a transgenic mouse model of AD, expressing two mutations in the human APP, as well as two human PS1 mutations knocked-in into the mouse PS1 gene in a homozygous (ho) manner. Although the hippocampus is significantly affected in these mice, very little is known about the effects of these mutations on selective neuronal populations and plaque load in the frontal cortex. In this study, cytoarchitectural changes were characterized using high precision design-based stereology to evaluate plaque load, total neuron numbers, as well as total numbers of parvalbumin- (PV) and calretinin- (CR) immunoreactive (ir) neurons in the frontal cortex of 2- and 10-month-old APP/PS1KI mice. The frontal cortex was divided into two subfields: layers II–IV and layers V–VI, the latter of which showed substantially more extracellular amyloid-beta aggregates. We found a 34% neuron loss in layers V–VI in the frontal cortex of 10-month-old APP/PS1KI mice compared to 2-month-old, while there was no change in PV- and CR-ir neurons in these mice. In addition, the plaque load in layers V–VI of 10-month-old APP/PS1KI mice was only 11% and did not fully account for the extent of neuronal loss. Interestingly, an increase was found in the total number of PV-ir neurons in all frontal cortical layers of single transgenic APP mice and in layers II–IV of single transgenic PS1ho mice between 2 and 10 months of age. In conclusion, the APP/PS1KI mice provide novel insights into the regional selective vulnerability in the frontal cortex during AD that, together with previous findings in the hippocampus, are remarkably similar to the human situation.

Cofilin-1 in the podocyte: a molecular switch for actin dynamics

Studies by Garg et al. and Ashworth et al. investigated the functional relevance of a key regulatory protein, cofilin-1, for podocyte actin dynamics (Ashworth et al. in PLos One 5:e12626, 2010; Garg et al. in J Biol Chem 285:22676-22688, 2010). Using different model organisms (zebrafish or transgenic mice), both groups observed a collapse of the glomerular filtration barrier upon inactivation of cofilin-1. In elegant biochemical studies, Garg et al. established that cofilin-1 activity is regulated by nephrin, which is part of the slit diaphragm complex. Two feedback loops stabilize cofilin-1 in the phosphorylated versus dephosphorylated state. The novel findings render cofilin-1 activity as potential diagnostic marker for pathological changes in the podocyte cytoarchitecture.

Ameliorative effect of NAP on laser-induced retinal damage

NAP is the smallest active element of activity-dependent neuroprotective protein (ADNP) in the non-myelinated neural tissue. This study evaluated the neuroprotective effect of NAP in reducing the spread of laser-induced retinal damage in rat. Laser lesions were created in 72 DA pigmented rats. Two groups were treated by one intravenous or intravitreal injection of NAP immediately after exposure to laser. Two control groups were similarly administered saline injection. Histological and morphometrical evaluations of the lesions were preformed 3, 20 and 60 days after photocoagulation. After intravitreal treatment with NAP, a significant reduction in the diameter of the laser-induced lesions was found 3 days after photocoagulation (p < 0.001) but not after 20 and 60 days while the systemic treatment significantly reduced lesion diameter 20 and 60 days after photocoagulation (p = 0.001). Significant difference in photoreceptor cell loss was found in eyes treated intravitreally only 3 days after photocoagulation (p = 0.002). In the systemically treated animals such effect was found only after 20 and 60 days (p < 0.001). Treatment with NAP ameliorates laser-induced retinal lesions. Intravitreal treatment had an early short-term effect while the effect of systemic administration was delayed and prolonged. This treatment may be of clinical significance in reducing laser-induced retinal injuries in humans.

Studying Brain &amp; Behavior in Schizophrenia: Multivariate Surface Analysis of Hippocampus

Studying Brain &amp; Behavior in Schizophrenia: Multivariate Surface Analysis of Hippocampus

Treatment with levothyroxine in early maternal subclinical hypothyroidism may improve the neural development of the progeny

Objective To investigate the effect of treatment with levothyroxine in early maternal subclinical hypothyroidism （SCH） on the neural development of the progeny. Methods 75 thyroidectomized female Wistar rats were divided randomly into groups of hypothyroidism （CH）, SCH, SCH treated with levothyroxine at embryonic day 10 （E10）, E13, and E17. There were 15 sham operated controls. Body weight,thyroid function, and the development of progeny by morris water maze, immunohistochemistry, and Nissl＇s staining of progeny were made. Results Pups from SCH or CH group had significantly lower body weight than euthyroid group （ P〈0. 05 ）. Pups from E10, E13 or E17 groups had normal body weight compared to pups of control （P〉0.05）. The levels of TSH and total T4（ TT4 ） of all pups were normal. The mean latencies were longer in pups from CH, SCH, and E17 group than the control （P〈0.05）. The mean escape latencies did not differ between the control and E10 group pups and between the control and E13 pups （P〉0.05）. There were changes in the cytoarchitecture of the barrel cortex and of the hippocampus （ toluidine blue-stained sections） in CH, SCH, and E17 pups. The barrel cortex of E10 or E13 pups was similar to that of control pups. The distribution of BrdUlabeled cells was more widespread in CH, SCH, and E17 pups than in control, E10, and E13 progeny.Conclusion Maternal SCH disturbs learning and memory performances, cytoarchitecture and cell migration of the pups. Treatment with levothyroxine in early maternal SCH before E13 improves the cell migration in the developing brain of the progeny. Key words: Subclinical hypothyroidism; Levothyroxine; Progeny; Neural development

Structural abnormalities revealed by magnetic resonance imaging in rats prenatally exposed to methylazoxymethanol acetate parallel cerebral pathology in schizophrenia

Schizophrenia is a highly familial, neurodevelopmental disorder that is associated with several neuropsychiatric, psychological, and neuropathological features. Although pharmacological animal models of dopaminergic and glutamatergic dysfunction have helped advance our understanding of the disease biology, there is a clear need for translational models that capture the neuropathological and functional manifestations associated with the intermediate phenotype and the clinical illness. Neuroimaging of preclinical neurodevelopmental approaches such as methylazoxymethanol acetate (MAM) exposure may afford a powerful translational tool to establish endpoints with greater congruency across animals and humans. Using in vivo volumetric magnetic resonance imaging (MRI), manganese-enhanced MRI, and diffusion tensor imaging (DTI), we investigated morphological and cytoarchitectural changes of brain structures in MAM-exposed rats, a neurodevelopmental model of schizophrenia. Compared to saline-exposed controls, MAM-exposed rats showed significant enlargement of lateral and third ventricles as well as reduced hippocampal volumes, which is consistent with findings observed in schizophrenia. In addition, DTI revealed that diffusion fractional anisotropy retrieved from corpus callosum and cingulum were significantly decreased in MAM-exposed rats, suggesting that demyelination occurred in these white-matter fiber tracts. Imaging findings were confirmed by conducting histological analysis using hematoxylin and eosin and Luxol fast blue stainings. In summary, structural abnormalities resulting from a MAM environmental challenge parallel cerebral pathology observed in schizophrenia. The MAM model incorporating noninvasive imaging techniques may therefore serve as an improved translational research tool for assessing new treatments for schizophrenia.

Pancreatic intraepithelial neoplasia – can we detect early pancreatic cancer?

Haugk B (2010) Histopathology 57, 503-514 Pancreatic intraepithelial neoplasia - can we detect early pancreatic cancer? Pancreatic cancer is one of the most lethal cancers, with an incidence equalling mortality. Pancreatic cancer is a heterogeneous group in which pancreatic ductal adenocarcinoma (PDAC) is the most common. It is now established that PDAC develops through stepwise progression from precursor lesions. Detection and treatment of these precursor lesions would allow curative treatment. Three precursor lesions for PDAC have been identified. Two of these - mucinous cystic neoplasms (MCNs) and intraductal papillary mucinous neoplasms (IPMNs) - are rare, radiologically detectable, cystic precursor lesions which can be cured if treated at the preinvasive stage. The third and most common precursor lesion has recently been defined as pancreatic intraepithelial neoplasia (PanIN). PanINs are microscopic lesions with no clinical correlate. They display a spectrum of cyto-architectural changes (PanIN-1, PanIN-2 and PanIN-3) mirrored in an increasing accumulation of molecular genetic changes, with PanIN-3 sharing many of the alterations with PDAC. Great advances in the understanding of pancreatic carcinogenesis have opened avenues for diagnosis and chemoprevention. However, access to the pancreas is limited, molecular tests are at the early stages and too little is known about the natural history of early PanINs to justify resection. Currently, screening focuses upon high-risk individuals only.

Thinning, movement, and volume loss of residual cortical tissue occurs after stroke in the adult rat as identified by histological and magnetic resonance imaging analysis

Plasticity of residual cortical tissue has been identified as an important mediator of functional post-stroke recovery. Many studies have been directed toward describing biochemical, electrophysiological, and cytoarchitectural changes in residual cortex and correlating them with functional changes. Additionally, after neonatal stroke the thickness of residual tissue can change, the tissue can move, and tissue can fill in the stroke core. The purpose of the present study was to systematically investigate and document possible gross morphological changes in peri-infarct tissue after forelimb motor cortex stroke in the adult rat. Rats received a unilateral forelimb motor cortex stroke of equivalent size by pial strip devascularization or photothrombotic occlusion and were then examined using histology or magnetic resonance imaging (MRI) at 1 h, 1, 3, 7, 14, or 31 days post-stroke. Middle cerebral artery occlusion was used as a control stroke procedure. Decreases in cortical thickness, volume, and neural density were found to extend far beyond the stroke infarct and included most of the sensorimotor regions of the stroke and intact hemispheres. Movement of residual tissue towards the infarct was observed and confirmed using anatomical markers placed in intact cortical tissue at the time of stroke induction. The results are discussed in relation to the idea that extensive time-dependent morphological changes that occur in residual tissue must be considered when evaluating plasticity-related cortical changes associated with post-stroke recovery of function.

A comparison of the tissue response to chronically implanted Parylene-C-coated and uncoated planar silicon microelectrode arrays in rat cortex

In this study we employed a quantitative immunohistochemical approach to compare the brain tissue response to planar silicon microelectrode arrays that were conformally coated with Parylene-C to uncoated controls at 2, 4, and 12 weeks following implantation into the cortex of adult male Sprague-Dawley rats. We did not find any difference in the relative intensity or the spatial distribution of neuronal or glial markers over the indwelling period, even though Parylene-C-coated substrates supported significantly less cell attachment, indicating that the foreign body response to planar silicon microelectrode arrays has little to do with the composition or decomposition of the silicon electrode. Moreover, our results suggest that changes in microelectrode surface chemistry do not have a strong influence on the cytoarchitectural changes that accompany the brain foreign body response to planar silicon microelectrode arrays. Our quantitative comparison over the indwelling period does not support progressive increases in astrocyte encapsulation and/or progressive neuronal loss in the recording zone as dominant failure mechanisms of the type of chronic recording device. Finally, we found evidence of two potentially new failure mechanisms that were associated with CD68 immunoreactivity including demyelination of adjacent neurons and BBB breakdown surrounding implanted electrodes at long indwelling times.

Cocaine causes deficits in radial migration and alters the distribution of glutamate and GABA neurons in the developing rat cerebral cortex.

Prenatal cocaine exposure induces cytoarchitectural changes in the embryonic neocortex; however, the biological mechanisms and type of cortical neurons involved in these changes are not known. Previously, we found that neural progenitor proliferation in the neocortical ventricular zone (VZ) is inhibited by cocaine; here, we examine the changes in cortical neurogenesis and migration of glutamate and GABA neurons induced by prenatal cocaine exposure. Pregnant rats received 20 mg/kg of cocaine intraperitoneally twice at an interval of 12 h during three periods of neocortical neurogenesis. Neocortical area and distribution of developing neurons were examined by counting Tuj1+, glutamate+, or GABA+ cells in different areas of the cerebral cortex. Cocaine decreased neocortical area by reducing the size of the Tuj1+ layer, but only when administered during early periods of neocortical neurogenesis. The number of glutamatergic neurons was increased in the VZ but was decreased in the outer cortical laminae. Although the number of GABA+ neurons in the VZ of both the neocortex and ganglionic eminences was unchanged, GABA+ cells decreased in all other neocortical laminae. Tangential migration of GABA+ cells was also disrupted by cocaine. These findings suggest that in utero cocaine exposure disturbs radial migration of neocortical neurons, possibly because of decreased radial glia guiding support through enhanced differentiation of neocortical VZ progenitors. Cocaine interrupts radial migration of both glutamatergic and GABAergic neurons within the neocortex, in addition to the tangential migration of GABAergic neurons from the subcortical telecephalon. This may result in abnormal neocortical cytoarchitecture and concomitant adverse functional effects.

Reproductive toxicity of sodium valproate in male rats

Objectives:To assess the effects of sodium valproate on rat sperm morphology, sperm count, motility, and histopathological changes in testis.Materials and Methods:Male Wistar rats (12 week old) were treated with sodium valpraote and sacrificed at the end of 2nd, 4th, 5th, 7th, 10th and 15th week after the last exposure to sodium valproate. Epididymal sperm count, sperm motility, sperm morphology, and histopathology of testes were analyzed.Results:Sperm count and sperm motility were decreased significantly by sodium valproate. The percentage of abnormal sperms increased in a dose-dependent manner. A histopathological study revealed that sodium valproate had caused sloughing of epithelial cells in testes.Conclusion:Sodium valproate causes reversible change in sperm motility, sperm count, morphology, and cytoarchitecture of testes.

Antihypertensive therapy in patients with liver diseases. Can we decrease the risk of liver fibrosis by affecting renin-angiotensin system?

The paper summarizes data on antihypertensive treatment in hypertensive patients with concomitant hepatic diseases when inflammation and fibrosis result in liver cytoarchitecture changes. Rennin-angiotensin-aldosterone system blockade enables to reduce tissue damage and fibrosis development regardless of blood pressure decrease. The majority of hypertensive patients requiring combination antihypertensive therapy can benefit in achieving target blood pressure faster, decreasing side effects risk, and in improving liver function. Key words: hypertension, liver diseases, rennin-angiotensin-aldosterone system blockade.

Coordinate Gα13 and Wnt6-β-catenin signaling in F9 embryonal carcinoma cells is required for primitive endoderm differentiation

The mouse F9 embryonal carcinoma cell line is ideally suited to study the epithelial-to-mesenchymal transition accompanying the differentiation of primitive to parietal extraembryonic endoderm. In F9 cells, the application of exogenous agents including retinoic acid or activation of signal transduction cascades downstream of G-proteins triggers widespread changes in gene expression and leads to the formation of primitive endoderm. The epithelial-to-mesenchymal transition is completed and parietal endoderm develops as of result of increasing PKA activity in primitive endoderm cells. Expression of a constitutively active form of Galpha13(Q226L) is sufficient to induce F9 cells into parietal endoderm and a model is emerging that a signaling axis linking G-protein signaling to RhoA and the ERM protein moesin is required for differentiation. In this study, we found that expression of either p115RhoGEF or a constitutively active, GTPase-deficient form of RhoA(L63) promoted primitive, but not parietal, endoderm formation. The overexpression of Galpha13(Q226L) or p115RhoGEF, but not Rho(L63), caused beta-catenin to translocate to the nucleus. Surprisingly, the stimulation of the Wnt-beta-catenin pathway was accompanied by nuclear beta-catenin and primitive endoderm formation, even when a dominant negative was used to block the signaling axis at the level of p115RhoGEF or when ROCK activity was inhibited using the pharmacological agent Y-27632. Together, results indicate that the coordinate signaling by two independent pathways, one involving canonical Wnt-beta-catenin activation of target genes and the other with Galpha13 signaling to ERM proteins to modulate cytoarchitectural changes, is required during the retinoic acid induced differentiation of F9 cells to primitive endoderm.

Impact of Growth Hormone (GH) Deficiency and GH Replacement upon Thymus Function in Adult Patients

BackgroundDespite age-related adipose involution, T cell generation in the thymus (thymopoiesis) is maintained beyond puberty in adults. In rodents, growth hormone (GH), insulin-like growth factor-1 (IGF-1), and GH secretagogues reverse age-related changes in thymus cytoarchitecture and increase thymopoiesis. GH administration also enhances thymic mass and function in HIV-infected patients. Until now, thymic function has not been investigated in adult GH deficiency (AGHD). The objective of this clinical study was to evaluate thymic function in AGHD, as well as the repercussion upon thymopoiesis of GH treatment for restoration of GH/IGF-1 physiological levels.Methodology/Principal FindingsTwenty-two patients with documented AGHD were enrolled in this study. The following parameters were measured: plasma IGF-1 concentrations, signal-joint T-cell receptor excision circle (sjTREC) frequency, and sj/β TREC ratio. Analyses were performed at three time points: firstly on GH treatment at maintenance dose, secondly one month after GH withdrawal, and thirdly one month after GH resumption. After 1-month interruption of GH treatment, both plasma IGF-1 concentrations and sjTREC frequency were decreased (p<0.001). Decreases in IGF-1 and sjTREC levels were correlated (r = 0.61, p<0.01). There was also a decrease in intrathymic T cell proliferation as indicated by the reduced sj/β TREC ratio (p<0.01). One month after reintroduction of GH treatment, IGF-1 concentration and sjTREC frequency regained a level equivalent to the one before GH withdrawal. The sj/β TREC ratio also increased with GH resumption, but did not return to the level measured before GH withdrawal.ConclusionsIn patients with AGHD under GH treatment, GH withdrawal decreases thymic T cell output, as well as intrathymic T cell proliferation. These parameters of thymus function are completely or partially restored one month after GH resumption. These data indicate that the functional integrity of the somatotrope GH/IGF-1 axis is important for the maintenance of a normal thymus function in human adults.Trial RegistrationClinicalTrials.gov NTC00601419

QLT0267, a small molecule inhibitor targeting integrin-linked kinase (ILK), and docetaxel can combine to produce synergistic interactions linked to enhanced cytotoxicity, reductions in P-AKT levels, altered F-actin architecture and improved treatment outcomes in an orthotopic breast cancer model

IntroductionSubstantial preclinical evidence has indicated that inhibition of integrin linked-kinase (ILK) correlates with cytotoxic/cytostatic cellular effects, delayed tumor growth in animal models of cancer, and inhibition of angiogenesis. Widely anticipated to represent a very promising therapeutic target in several cancer indications, it is increasingly evident that optimal therapeutic benefits obtained using ILK targeting strategies will only be achieved in combination settings. The purpose of this study was to investigate the therapeutic potential of the ILK small molecule inhibitor, QLT0267 (267), alone or in combination with chemotherapies commonly used to treat breast cancer patients.MethodsA single end-point metabolic assay was used as an initial screen for 267 interactions with selected chemotherapeutic agents. These in vitro assays were completed with seven breast cancer cell lines including several which over-expressed human epidermal growth factor receptor 2 (Her2). One agent, docetaxel (Dt), consistently produced synergistic interactions when combined with 267. Dt/267 interactions were further characterized by measuring therapeutic endpoints linked to phosphorylated protein kinase B (P-AKT) suppression, inhibition of vascular endothelial growth factor (VEGF) secretion and changes in cytoarchitecture. In vivo efficacy studies were completed in mice bearing orthotopic xenografts where tumor growth was assessed by bioluminescence and calliper methods.ResultsThe combination of 267 and Dt resulted in increased cytotoxic activity, as determined using an assay of metabolic activity. Combinations of cisplatin, doxorubicin, vinorelbine, paclitaxel, and trastuzumab produced antagonistic interactions. Further endpoint analysis in cell lines with low Her2 levels revealed that the 267/Dt combinations resulted in: a three-fold decrease in concentration (dose) of 267 required to achieve 50% inhibition of P-AKT; and a dramatic disruption of normal filamentous-actin cellular architecture. In contrast to Her2-positive cell lines, three-fold higher concentrations of 267 were required to achieve 50% inhibition of P-AKT when the drug was used in combination with Dt. In vivo studies focusing on low Her2-expressing breast cancer cells (LCC6) implanted orthotopically demonstrated that treatment with 267/Dt engendered improved therapeutic effects when compared with mice treated with either agent alone.ConclusionsThe findings indicate that the 267/Dt drug combination confers increased (synergistic) therapeutic efficacy towards human breast cancer cells that express low levels of Her2.