Dissecting population and single-cell heterogeneity in response to anti-cancer drugs

Die Wirkung von niedermolekularen Krebsmedikamenten wird normalerweise mittels zell-basierten Analysen getestet, um die Wirksamkeit zu optimieren, Erkenntnisse über Zellwachstum und -tod zu gewinnen, sowie Faktoren zu identifizieren, die Resistenz und Sensitivität beeinflussen. Um die Wirkmechanismen von Therapeutika zu verstehen, müssen die arzneimittelbedingten Veränderungen auch hinsichtlich des intrazellulären Zustandes (die Inhibition der Onkogen-Signalwege, Veränderungen in dem Zellzyklus und Induktion von Seneszenz oder Apoptose) charakterisiert werden. Naheliegende konventionelle Einzelparameter Screening Techniken, wie sie in der Arzneimittelforschung genutzt werden, sind nicht im Stande diese Komplexität methodisch zu bewerten. Deshalb sind Multiplex Methoden erforderlich, um den Dosis-Wirkungs-Verlauf von multiplen molekularen Signalen und zellulären Phänotypen zu erfassen. In dieser These behaupten wir, dass hochauflösende und Hochdurchsatz-Mikroskopie für diesen Verwendungszweck ideal ist, da nicht nur Aspekte wie Phänotyp- und Ziel-basierende Ansätze, sondern auch molekulare Details der medikamentös-induzierten Phänotypen der einzelnen Zellen einbezogen werden können. Die Analyse der phänotypischen Dosis-Wirkungs-Kurven von neun Kinase-Inhibitoren, gemessen mittels Hochdurchsatz-Mikroskopie, zeigt systematische Parallelen, aber auch Variationen zwischen den verschiedenen Medikamenten. Interessanterweise verursacht die Inhibition von mTOR einen dosisabhängigen Anstieg der Phosphorylation von ERK1/2, der die Wirkung verringern könnte. Obwohl die durchschnittlichen Messungen der Dosiswirkung zweifellos nützlich sind, um das Verhalten der gesamten Zellenpopulation auf die entsprechenden Medikamente zu erforschen, wird die Einzel-Zell-Analyse zunehmend als Möglichkeit für die Erforschung von natürlichen und induzierten Veränderungen in der Zellphysiologie anerkannt. Wir überwachten die Reaktion auf vier Kinase-Inhibitoren, die auf den frühen Signalweg wirken. Wir verwendeten dazu eine neuartige “zyklische Immunfluoreszenz” Methode (CycIF), die fähig ist bis zu 30 Kanäle gleichzeitig abzubilden. Zur Datenanalyse wurden bereits bestehende Hilfsmittel wie k-means-Algorithmus, viSNE- und Wanderlust-Algorithmus verwendet. Wir waren in der Lage verschiedene Abschnitte des Zellzyklus mit der Wanderlust-Zeitschiene darzustellen. Mittels viSNE-Algorithmus konnten wir zeigen, dass verschiedene Medikamente zur Entstehung von eigenständigen Subpopulationen führen. Folglich offenbart das “Multiplex-Single-Cell-Imaging” nützliche Einblicke in die Wirkmechanismen von Medikamenten und Zell-zu-Zell-Variabilität

FRET-Based Calcium Imaging: A Tool for High-Throughput/Content Phenotypic Drug Screening in Alzheimer Disease

Perturbed intracellular store calcium homeostasis is suggested to play a major role in the pathophysiology of Alzheimer disease (AD). A number of mechanisms have been suggested to underlie the impairment of endoplasmic reticulum calcium homeostasis associated with familial AD-linked presenilin 1 mutations (FAD-PS1). Without aiming at specifically targeting any of those pathophysiological mechanisms in particular, we rather performed a high-throughput phenotypic screen to identify compounds that can reverse the exaggerated agonist-evoked endoplasmic reticulum calcium release phenotype in HEK293 cells expressing FAD-PS1. For that purpose, we developed a fully automated high-throughput calcium imaging assay using a fluorescence resonance energy transfer-based calcium indicator at single-cell resolution. This novel robust assay offers a number of advantages compared with the conventional calcium measurement screening technologies. The assay was employed in a large-scale screen with a library of diverse compounds comprising 20,000 low-molecular-weight molecules, which resulted in the identification of 52 primary hits and 4 lead structures. In a secondary assay, several hits were found to alter the amyloid (A) production. In view of the recent failure of AD drug candidates identified by target-based approaches, such a phenotypic drug discovery paradigm may present an attractive alternative for the identification of novel AD therapeutics

Identification of tetrahydrocarbazoles as novel multifactorial drug candidates for treatment of Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder and the most frequent cause of dementia. To date, there are only a few approved drugs for AD, which show little or no effect on disease progression. Impaired intracellular calcium homeostasis is believed to occur early in the cascade of events leading to AD. Here, we examined the possibility of normalizing the disrupted calcium homeostasis in the endoplasmic reticulum (ER) store as an innovative approach for AD drug discovery. High-throughput screening of a small-molecule compound library led to the identification of tetrahydrocarbazoles, a novel multifactorial class of compounds that can normalize the impaired ER calcium homeostasis. We found that the tetrahydrocarbazole lead structure, first, dampens the enhanced calcium release from ER in HEK293 cells expressing familial Alzheimer's disease (FAD)-linked presenilin 1 mutations. Second, the lead structure also improves mitochondrial function, measured by increased mitochondrial membrane potential. Third, the same lead structure also attenuates the production of amyloid-beta (A beta) peptides by decreasing the cleavage of amyloid precursor protein (APP) by beta-secretase, without notably affecting alpha- and gamma-secretase cleavage activities. Considering the beneficial effects of tetrahydrocarbazoles addressing three key pathological aspects of AD, these compounds hold promise for the development of potentially effective AD drug candidates

RASSF1A independence and early galectin-1 upregulation in PIK3CA-induced hepatocarcinogenesis: new therapeutic venues

Aberrant activation of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR and Ras/mitogen-activated protein kinase (MAPK) pathways is a hallmark of hepatocarcinogenesis. In a subset of hepatocellular carcinomas (HCCs), PI3K/AKT/mTOR signaling dysregulation depends on phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations, while RAS/MAPK activation is partly attributed to promoter methylation of the tumor suppressor Ras association domain-containing protein 1 (RASSF1A). To evaluate a possible cocarcinogenic effect of PIK3CA activation and RASSF1A knockout, plasmids expressing oncogenic forms of PIK3CA (E545K or H1047R mutants) were delivered to the liver of RASSF1A knockout and wild-type mice by hydrodynamic tail vein injection combined with sleeping beauty-mediated somatic integration. Transfection of either PIK3CA E545K or H1047R mutants sufficed to induce HCCs in mice irrespective of RASSF1A mutational background. The related tumors displayed a lipogenic phenotype with upregulation of fatty acid synthase and stearoyl-CoA desaturase-1 (SCD1). Galectin-1, which was commonly upregulated in preneoplastic lesions and tumors, emerged as a regulator of SCD1. Co-inhibitory treatment with PIK3CA inhibitors and the galectin-1 inhibitor OTX008 resulted in synergistic cytotoxicity in human HCC cell lines, suggesting novel therapeutic venues

Identification of tetrahydrocarbazoles as novel multifactorial drug candidates for treatment of Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder and the most frequent cause of dementia. To date, there are only a few approved drugs for AD, which show little or no effect on disease progression. Impaired intracellular calcium homeostasis is believed to occur early in the cascade of events leading to AD. Here, we examined the possibility of normalizing the disrupted calcium homeostasis in the endoplasmic reticulum (ER) store as an innovative approach for AD drug discovery. High-throughput screening of a small-molecule compound library led to the identification of tetrahydrocarbazoles, a novel multifactorial class of compounds that can normalize the impaired ER calcium homeostasis. We found that the tetrahydrocarbazole lead structure, first, dampens the enhanced calcium release from ER in HEK293 cells expressing familial Alzheimer's disease (FAD)-linked presenilin 1 mutations. Second, the lead structure also improves mitochondrial function, measured by increased mitochondrial membrane potential. Third, the same lead structure also attenuates the production of amyloid-beta (A beta) peptides by decreasing the cleavage of amyloid precursor protein (APP) by beta-secretase, without notably affecting alpha- and gamma-secretase cleavage activities. Considering the beneficial effects of tetrahydrocarbazoles addressing three key pathological aspects of AD, these compounds hold promise for the development of potentially effective AD drug candidates

Upregulation of CRABP1 in human neuroblastoma cells overproducing the Alzheimer-typical Aβ42 reduces their differentiation potential

<p>Abstract</p> <p>Background</p> <p>Alzheimer's disease (AD) is characterized by neurodegeneration and changes in cellular processes, including neurogenesis. Proteolytic processing of the amyloid precursor protein (APP) plays a central role in AD. Owing to varying APP processing, several β-amyloid peptides (Aβ) are generated. In contrast to the form with 40 amino acids (Aβ₄₀), the variant with 42 amino acids (Aβ₄₂) is thought to be the pathogenic form triggering the pathological cascade in AD. While total-Aβ effects have been studied extensively, little is known about specific genome-wide effects triggered by Aβ₄₂or Aβ₄₀derived from their direct precursor C99.</p> <p>Methods</p> <p>A combined transcriptomics/proteomics analysis was performed to measure the effects of intracellularly generated Aβ peptides in human neuroblastoma cells. Data was validated by real-time polymerase chain reaction (real-time PCR) and a functional validation was carried out using RNA interference.</p> <p>Results</p> <p>Here we studied the transcriptomic and proteomic responses to increased or decreased Aβ₄₂and Aβ₄₀levels generated in human neuroblastoma cells. Genome-wide expression profiles (Affymetrix) and proteomic approaches were combined to analyze the cellular response to the changed Aβ₄₂- and Aβ₄₀-levels. The cells responded to this challenge with significant changes in their expression pattern. We identified several dysregulated genes and proteins, but only the cellular retinoic acid binding protein 1 (CRABP1) was up-regulated exclusively in cells expressing an increased Aβ₄₂/Aβ₄₀ratio. This consequently reduced all-trans retinoic acid (RA)-induced differentiation, validated by CRABP1 knock down, which led to recovery of the cellular response to RA treatment and cellular sprouting under physiological RA concentrations. Importantly, this effect was specific to the AD typical increase in the Aβ₄₂/Aβ₄₀ratio, whereas a decreased ratio did not result in up-regulation of CRABP1.</p> <p>Conclusion</p> <p>We conclude that increasing the Aβ₄₂/Aβ₄₀ratio up-regulates CRABP1, which in turn reduces the differentiation potential of the human neuroblastoma cell line SH-SY5Y, but increases cell proliferation. This work might contribute to the better understanding of AD neurogenesis, currently a controversial topic.</p