Resenzitizace leukemických a lymfomových buněk k TRAILem indukované apoptóze

Apoptóza představuje přirozenou bariéru proti rozvoji nádorových onemocnění a rezistence nádorových buněk k apoptóze je jednou ze základních vlastností získaných během tumorigeneze. Protože většina v současné době používaných cytostatik vyvolává zánik nádorových buněk aktivací vnitřní apoptotické dráhy, je porucha aktivace vnitřní apoptotické dráhy spojena se selháním terapie. Cílená aktivace zevní apoptotické dráhy umožňuje indukci apoptózy i u nádorových buněk rezistentních k cytostatikům. TRAIL je cytokin patřící do rodiny TNFα, který specificky indukuje zevní apoptotickou dráhu v nádorových buňkách a je netoxický vůči normálním buňkám. Vzhledem k těmto vlastnostem představuje TRAIL slibnou protinádorovou látku. Velká část primárních nádorů je však vůči TRAILu rezistentní. Ve snaze o překonání rezistence nádorových buněk na TRAIL byla identifikována celá řada látek, které zvyšují citlivost nádorových buněk na TRAIL. Předchozí studie ukazují, že roskovitin, inhibitor cyklin-dependentních kináz, zvyšuje citlivost solidních nádorů na TRAIL. V této práci jsme studovali citlivost různých hematologických malignit k TRAILem indukované apoptóze. Následně jsme zjišťovali schopnost roskovitinu zvýšit citlivost leukemických a lymfomových buněk na TRAIL in vitro a in vivo na myším modelu lidského lymfomu. Nakonec...Apoptosis serves as a natural barrier to cancer development, and the resistance to apoptosis represents one of the key capabilities acquired during tumor development or progression. Impairment of the intrinsic apoptotic pathway exemplifies one of the established mechanisms of constitutive or acquired drug resistance. As most of the currently used cytotoxic drugs initiate tumor cell death by direct or indirect triggering of the intrinsic apoptotic pathway, impairment of the intrinsic pathway is associated with therapy failure. Targeting of the death receptors, however, enables induction of apoptosis even in the chemotherapy resistant cancer cells. TRAIL is a death ligand belonging to the TNFα superfamily that specifically kills tumor cells while sparing healthy tissues. Much enthusiasm has been generated for TRAIL as a highly promising targeted anti-cancer agent. However, many primary tumors have been shown to be TRAIL resistant. In attempt to overcome such an intrinsic TRAIL resistance a wide array of agents have been shown to sensitize tumor cells to TRAIL. Previous studies reported that roscovitine, a cyclin-dependent kinase inhibitor, sensitized various solid cancer cells to TRAIL. In this study we analyzed the sensitivity of diverse hematologic malignancies to TRAIL-induced apoptosis and measured the...Institute of Pathological Physiology First Faculty of Medicine Charles University in PragueÚstav patologické fyziologie 1. LF UK v PrazeFirst Faculty of Medicine1. lékařská fakult

Sustained proliferation in cancer: mechanisms and novel therapeutic targets

Proliferation is an important part of cancer development and progression. This is manifest by altered expression and/or activity of cell cycle related proteins. Constitutive activation of many signal transduction pathways also stimulates cell growth. Early steps in tumor development are associated with a fibrogenic response and the development of a hypoxic environment which favors the survival and proliferation of cancer stem cells. Part of the survival strategy of cancer stem cells may manifested by alterations in cell metabolism. Once tumors appear, growth and metastasis may be supported by overproduction of appropriate hormones (in hormonally dependent cancers), by promoting angiogenesis, by undergoing epithelial to mesenchymal transition, by triggering autophagy, and by taking cues from surrounding stromal cells. A number of natural compounds (e.g., curcumin, resveratrol, indole-3-carbinol, brassinin, sulforaphane, epigallocatechin-3-gallate, genistein, ellagitannins, lycopene and quercetin) have been found to inhibit one or more pathways that contribute to proliferation (e.g., hypoxia inducible factor 1, nuclear factor kappa B, phosphoinositide 3 kinase/Akt, insulin-like growth factor receptor 1, Wnt, cell cycle associated proteins, as well as androgen and estrogen receptor signaling). These data, in combination with bioinformatics analyses, will be very important for identifying signaling pathways and molecular targets that may provide early diagnostic markers and/or critical targets for the development of new drugs or drug combinations that block tumor formation and progression

Antitumoral Properties of Natural Products

Cancer is one of the major causes of death worldwide. It is a multifactorial heterogeneous disease characterized by the transformation of normal cells into malignant cells, which acquire an uncontrolled growth, immortality, invasiveness, and ability to form distant metastasis. Natural bioactive molecules may interfere with these processes and inhibit the carcinogenesis process. In this book, new molecules and extracts, mainly derived from plants, have been described as being able to alter tumor cell behavior and target several abnormal molecular pathways in cancer cells. Among different cancer cells, the more studied include those derived from glioblastoma, osteosarcoma, lung, breast and gastric cancer. These natural products could be an attractive source for the development of new preventative and therapeutic agents against cancer. They may be more selective and have weaker adverse effects compared to conventional chemotherapy drugs that are actually used for cancer treatment. Clinical trials are necessary to demonstrate whether the in vitro and in vivo animal data are reproduced in humans before the application of natural products in cancer prevention and treatment

Kanonické a nekanonické signální dráhy aktivované receptory pro ligand TRAIL v lidských buňkách

TRAIL ligand spouští apoptózu interakcí se svými dvěma pro-apoptotickými receptory TRAIL- R1 (DR4) a TRAIL-R2 (DR5). Jeho schopnost indukovat apoptózu nezávisle na statusu p53 a selektivně zabíjet rakovinné buňky in vitro a in vivo zapříčinila, že tento ligand patřící do rodiny cytokinů TNF se jevil atraktivním cílem výzkumu terapie nádorových onemocnění. Avšak rezistence mnoha primárních nádorových buněk, neuspokojivý výsledek klinických testů a nedávné studie, které ukazují protichůdné výsledky a to konkrétně, že TRAIL může za specifických podmínek podporovat místo toho, aby inhiboval progresi nádorů, vyvolaly v oblasti výzkumu signalizace indukované ligandem TRAIL nové množství otázek, které je potřeba zodpovědet. Ačkoliv jsou oba receptory DR4 a DR5 rovnoměrne exprimovány, různé typy nádorů vykazují preferenci pro jeden nebo druhý z receptorů. Relativní účast signalizace DR4 a DR5 indukované TRAIL ligandem je málo známá. Abychom analyzovali signalizaci ligandu TRAIL specifickou pro jednotlivé receptory, připravili jsme trimerizované varianty rekombinantního lidského ligandu TRAIL, značené pomocí Strep-tag, s vysokou afinitou pro receptory DR4 nebo DR5. Zjistila jsem, že narozdíl od pankreatických rakovinových buňek PANC-1 rezistentních k apoptóze indukované ligandem TRAIL, probíhá tvorba...TRAIL ligand can trigger apoptosis of permissive human cells via engagement of its two pro- apoptotic receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Its ability to induce apoptosis independently on p53 status and to selectively kill cancer cells in vitro and in vivo made this ligand an attractive target in cancer research. However, acquired resistance of primary cancer cells, unsatisfactory outcome of clinical trials and recent studies arguing that TRAIL might under specific conditions promote cancer progression, opened new plethora of questions, which need to be addressed. Though both receptors DR4 and DR5 are ubiquitously expressed, different types of tumours show preference for either of the receptors. The relative participation of DR4 and DR5 in TRAIL- induced signalling is still largely unknown. To analyse TRAIL receptor-specific signalling, I prepared Strep-tagged, trimerised variants of recombinant human TRAIL ligands with high affinity for either DR4 or DR5 receptor. Using these receptor-specific ligands, I examined a contribution of individual pro-apoptotic receptors to TRAIL-induced signalling pathways. I found that in TRAIL resistant colorectal HT-29 cells but not in pancreatic PANC-1 cancer cells, DISC formation and initial caspase-8 processing proceeded comparably in both DR4- and...Katedra genetiky a mikrobiologieDepartment of Genetics and MicrobiologyFaculty of SciencePřírodovědecká fakult

Carbon-Ion Irradiation Suppresses Migration and Invasiveness of Human Pancreatic Carcinoma Cells MIAPaCa-2 via Rac1 and RhoA Degradation

PurposeTo investigate the mechanisms underlying the inhibition of cancer cell migration and invasion by carbon (C)-ion irradiation.Methods and MaterialsHuman pancreatic cancer cells MIAPaCa-2, AsPC-1, and BxPC-3 were treated by x-ray (4 Gy) or C-ion (0.5, 1, 2, or 4 Gy) irradiation, and their migration and invasion were assessed 2 days later. The levels of guanosine triphosphate (GTP)-bound Rac1 and RhoA were determined by the active GTPase pull-down assay with or without a proteasome inhibitor, and the binding of E3 ubiquitin ligase to GTP-bound Rac1 was examined by immunoprecipitation.ResultsCarbon-ion irradiation reduced the levels of GTP-bound Rac1 and RhoA, 2 major regulators of cell motility, in MIAPaCa-2 cells and GTP-bound Rac1 in AsPC-1 and BxPC-3 cells. Proteasome inhibition reversed the effect, indicating that C-ion irradiation induced Rac1 and RhoA degradation via the ubiquitin (Ub)-proteasome pathway. E3 Ub ligase X-linked inhibitor of apoptosis protein (XIAP), which directly targets Rac1, was selectively induced in C-ion–irradiated MIAPaCa-2 cells and coprecipitated with GTP-bound Rac1 in C-ion–irradiated cells, which was associated with Rac1 ubiquitination. Cell migration and invasion reduced by C-ion radiation were restored by short interfering RNA–mediated XIAP knockdown, indicating that XIAP is involved in C-ion–induced inhibition of cell motility.ConclusionIn contrast to x-ray irradiation, C-ion treatment inhibited the activity of Rac1 and RhoA in MIAPaCa-2 cells and Rac1 in AsPC-1 and BxPC-3 cells via Ub-mediated proteasomal degradation, thereby blocking the motility of these pancreatic cancer cells

Apoptosis and mitotic slippage following drug intervention in leukaemia cells

PhDThe response of leukaemia cells to therapeutic agents includes cell cycle arrest and apoptosis. The former response is useful in retarding disease progression, but induction of the latter is essential for disease eradication. Cell death is often related to toxicity so reducing drug-concentration or sensitising target cells to apoptosis is desirable. The relationship between the cell cycle and cell death has been at the centre of recent investigation focusing on mechanisms of cell death that are not driven directly by apoptotic responses. These mechanisms included mitotic catastrophe and mitotic slippage. The K562 myeloid leukaemia cell line exhibits a combination of p53 negativity and carries the Bcr-Abl t (9:22) Philadelphia chromosome. Bcr-Abl is a powerful anti-apoptotic translocation and is the hallmark of chronic myeloid leukaemia (CML). The absence of p53-mediated apoptosis and the anti-apoptotic effects of Bcr-abl delays drug-induced cell death, leaving a window of opportunity to investigate the effects of different agents on leukaemia cells. My investigations show that when DNA-targeting agents are used against myeloid leukaemia cells, G2 cell cycle arrest and apoptosis do not occur together i.e. cell cycle arrest precludes cell death; cells may escape G2 arrest as a result of mitotic slippage. In contrast, when anti-mitotic agents are used, it is necessary to induce mitotic arrest to subsequently induce apoptosis; thus lower concentrations are more effective in inducing apoptosis than higher drug concentrations. Evidence is provided suggesting reduced concentrations of both genotoxic agents and anti-mitotic agents may share a common pathway in inducing cell death that is related to events at mitosis and I suggest that this pathway has potential for exploitation by new agents currently in clinical trials, such as UCN-01, Purvanol, Roscovitine and agents that target the passenger proteins, in reducing the concentration of more conventional agents required to kill the Bcr-Abl positive leukaemias

Targeting aberrant kinase activity in myeloid leukaemias

Acute myeloid leukaemia (AML) is the most common form of acute leukaemia in adults. Its treatment has remained largely unchanged for the past 30 years. Chronic myeloid leukaemia (CML) represents a tremendous success story in the era of targeted therapy but significant challenges remain including the development of drug resistance and disease persistence due to presence of CML stem cells. The Aurora family of kinases is essential for cell cycle regulation and their aberrant expression in cancer prompted the development of small molecules that selectively inhibit their activity. Chapter 2 of this thesis outlines the efficacy and mechanism of action of alisertib, a novel inhibitor of Aurora A kinase, in preclinical models of CML. Alisertib possessed equipotent activity against CML cells expressing unmutated and mutated forms of BCR-ABL. Notably, this agent retained high activity against the T315I and E255K BCR-ABL mutations, which confer the greatest degree of resistance to standard CML therapy. Chapter 3 explores the activity of alisertib in preclinical models of AML. Alisertib disrupted cell viability, diminished clonogenic survival, induced expression of the forkhead box O3 (FOXO3a) targets p27 and BCL-2 interacting mediator (BIM), and triggered apoptosis. A link between Aurora A expression and sensitivity to ara-C was established. Chapter 4 outlines the role of the proto-oncogene serine/threonine-protein (PIM) kinases in resistance to ara-C in AML. We report that the novel small molecule PIM kinase inhibitor SGI-1776 disrupted cell viability and induced apoptosis in AML. We establish a link between ara-C resistance and PIM over-expression. Finally, chapter 5 explores how the preclinical work outlined in this thesis may be translated into clinical studies that may lead to novel therapeutic approaches for patients with refractory myeloid leukaemia

Targeting Multiple Signal Transduction Pathways of SARS-CoV-2: Approaches to COVID-19 Therapeutic Candidates

Due to the complicated pathogenic pathways of coronavirus disease 2019 (COVID-19), related medicinal therapies have remained a clinical challenge. COVID-19 highlights the urgent need to develop mechanistic pathogenic pathways and effective agents for preventing/treating future epidemics. As a result, the destructive pathways of COVID-19 are in the line with clinical symptoms induced by severe acute coronary syndrome (SARS), including lung failure and pneumonia. Accordingly, revealing the exact signaling pathways, including inflammation, oxidative stress, apoptosis, and autophagy, as well as relative representative mediators such as tumor necrosis factor-α (TNF-α), nuclear factor erythroid 2-related factor 2 (Nrf2), Bax/caspases, and Beclin/LC3, respectively, will pave the road for combating COVID-19. Prevailing host factors and multiple steps of SARS-CoV-2 attachment/entry, replication, and assembly/release would be hopeful strategies against COVID-19. This is a comprehensive review of the destructive signaling pathways and host–pathogen interaction of SARS-CoV-2, as well as related therapeutic targets and treatment strategies, including potential natural products-based candidatesJ.E. gratefully acknowledges funding from CONICYT (PAI/ACADEMIA N°79160109)S