Main Cellular Target Research Articles

Modified (2′-deoxy)adenosines activate autophagy primarily through AMPK/ULK1-dependent pathway

Autophagy is a conserved self-digestion process, which governs regulated degradation of cellular components. Autophagy is upregulated upon energy shortage sensed by AMP-dependent protein kinase (AMPK). Autophagy activators might be contemplated as therapies for metabolic neurodegenerative diseases and obesity, as well as cancer, considering tumor-suppressive functions of autophagy. Among them, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr), a nucleoside precursor of the active phosphorylated AMP analog, is the most commonly used pharmacological modulator of AMPK activity, despite its multiple reported “off-target” effects. Here, we assessed the autophagy/mitophagy activation ability of a small set of (2′-deoxy)adenosine derivatives and analogs using a fluorescent reporter assay and immunoblotting analysis. The first two leader compounds, 7,8-dihydro-8-oxo-2′-deoxyadenosine and -adenosine, are nucleoside forms of major oxidative DNA and RNA lesions. The third, a derivative of inactive N6-methyladenosine with a metabolizable phosphate-masking group, exhibited the highest activity in the series. These compounds primarily contributed to the activation of AMPK and outperformed AICAr; however, retaining the activity in knockout cell lines for AMPK (ΔAMPK) and its upstream regulator SIRT1 (ΔSIRT1) suggests that AMPK is not a main cellular target. Overall, we confirmed the prospects of searching for autophagy activators among (2′-deoxy)adenosine derivatives and demonstrated the applicability of the phosphate-masking strategy for increasing their efficacy.

Chemoproteomics of Marine Natural Product Naamidine J Unveils CSE1L as a Therapeutic Target in Acute Lung Injury.

Acute lung injury is a devastating illness characterized by severe inflammation mediated by aberrant activation of macrophages, resulting in significant morbidity and mortality, highlighting the urgent need for novel pharmacological targets and drug candidates. In this study, we identified a novel target for regulating inflammation in macrophages and acute lung injury via chemical proteomics and genetics based on a marine alkaloid, naamidine J (NJ). The structures of NJ-related naamidine alkaloids were first confirmed or revised by a combination of quantum chemical calculations and X-ray diffraction analysis. NJ was found as a potential anti-inflammatory agent by screening our compound library, and CSE1L was identified by chemoproteomics as a main cellular target of NJ to inhibit inflammation in macrophages and protect against acute lung injury. Mechanistically, we demonstrated that NJ directly interacted with CSE1L on the sites of His745 and Phe903 and then inhibited the nuclear translocation and transcriptional activity of transcription factor SP1, thereby suppressing inflammation in macrophages and ameliorating acute lung injury. Taken together, these findings have uncovered a novel pharmacological target for the treatment of acute lung injury and have also provided a potential druggable pocket of CSE1L and a lead compound or an available chemical tool from marine sources for investigating CSE1L function and developing novel drug candidates against acute lung injury.

Abstract 2033: Rigosertib promotes anti-tumor activity of cancer cells via CETSA revealed novel targets and activates NLRP3-dependent inflammatory responses

Abstract Background: Rigosertib (ON 01910.Na) is considered a promising anticancer agent due to its ability to inhibit multiple disease signaling pathways, including RAS-MAPK signaling. Rigosertib is in a Phase 2 program in advanced squamous cell carcinoma complicating recessive dystrophic epidermolysis bullosa (RDEB-associated SCC) (NCT03786237, NCT04177498), and a Phase 2 trial in combination with pembrolizumab in patients with metastatic melanoma (NCT05764395). The aim of our study was to further elucidate the mechanism of action and to identify potential novel targets engaged by rigosertib. Methods: A variety of biochemical, molecular and cell biological assays were performed to characterize the binding of rigosertib to previously proposed targets and its effect on RAS-MAPK signaling and tumor cell survival. A mass spectrometry-based Cellular Thermal Shift Assay (CETSA-MS) was conducted to identify potential new protein targets of rigosertib. Time-lapse microscopy was used for detection of ROS levels and tubulin polymerization in rigosertib treated lung cancer cells. Bone Marrow Derived Macrophages (BMDM) collected from C57/BL6 mice and human lung cancer organoids were treated with rigosertib and the cell death was determined by flow cytometry. Results: Comparative CETSA profiling of rigosertib and colchicine, a classical tubulin depolymerizing agent, reveals unique targets of rigosertib in multiple tumor cell types. We identify two ROS-related proteins, ERO1A and NQO2 that potentially contribute to the induction of ROS-dependent JNK activation. Fluorescence live-cell imaging confirms the effect of ERO1A and NQO2 on rigosertib induced ROS generation. NEK7, a relevant protein for microtubule organization, is another potential target for rigosertib. NEK7 knock-down reveals reduced microtubule depolymerization in rigosertib treated lung cancer cells. While we confirm that rigosertib might affect microtubules at higher concentrations, the main cellular target(s) responsible for the induction of stress and JNK-mediated inhibition of RAS-RAF-MEK signaling needs to be further characterized. Rigosertib affects the tumor immune environment by activating NLRP3-dependent inflammatory responses and Caspase-1 to trigger IL-1β and IL-18 secretion in THP1 cells, as well as in human cancer organoids, and in wild-type, Casp1/11−/−, Asc−/− and Nlrp3−/− BMDMs. Conclusion: These findings indicate that rigosertib may represent an effective compound for inhibition of RAS-MAPK signaling through ROS-mediated JNK activation. The rigosertib-dependent mechanism for NLRP3 activation suggests a reprogramming of the tumor immune environment which may contribute to the synergistic effect seen with immune checkpoint inhibitors pre-clinically and clinically. Clinical trials in several difficult-to-treat cancers continue. Citation Format: Petros Kechagioglou, Camille Dupont, Hajime Yurugi, Alexey Chernobrovkin, Rossana Romero, Rebecca Tweedell, Thirumala-Devi Kanneganti, Stephen Cosenza, Steven M. Fruchtman, Krishnaraj Rajalingam. Rigosertib promotes anti-tumor activity of cancer cells via CETSA revealed novel targets and activates NLRP3-dependent inflammatory responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2033.

Abstract A020: CETSA profiling unveils novel targets engaged by anti-tumor drug rigosertib to inhibit RAS-MAPK signaling and trigger NLRP3 inflammasome activation

Abstract Background: The importance of styryl benzyl sulfones that induce G2/M cell cycle arrest and apoptosis in tumor cells has raised interest in cancer therapeutics. Rigosertib (ON 01910.Na) has been characterized as a disruptor of multiple signaling pathways including RAS-MAPK signaling through multiple mechanisms. The aim of our study was to further elucidate the mechanism of action and to identify potential novel targets engaged by rigosertib. Methods: Thermal shift, NanoBiT, in vitro tubulin polymerization and a variety of biochemical, molecular and cell biological assays were performed to characterize the binding of rigosertib to previously proposed targets and its effect on RAS-MAPK signaling and tumor cell survival. A mass spectrometry-based Cellular Thermal Shift Assay (CETSA-MS) that serves as a key tool to identify the targets in the entire proteome engaged by chemical ligands or small molecules of interest at physiological levels in cells, was conducted in multiple cell types to identify potential new protein targets of rigosertib. Results: Our data suggest that rigosertib exerts its inhibitory effect on RAS-MAPK signaling through ROS induced activation of JNK signaling. While we confirm that rigosertib might affect microtubules at higher concentrations, the main cellular target(s) responsible for the induction of stress and JNK-mediated inhibition of RAS-RAF-MEK signaling needs to be further characterized. By performing comparative CETSA profiling of rigosertib and colchicine, a classical anti mitotic drug that binds to soluble tubulin, we are able to identify unique targets of rigosertib in multiple tumor cell types. In particular, we identify two ROS-related proteins, ERO1A and NQO2 that potentially contribute to the induction of ROS-dependent JNK activation. NEK7, a relevant protein for microtubule organization and essential for NLRP3 inflammasome activation, is another potential target for rigosertib. Indeed, rigosertib activates NLRP3-dependent inflammatory responses via NEK7 and Caspase-1 to trigger IL-1β secretion. Conclusion: These results suggest that rigosertib may represent an effective compound for inhibition of RAS-MAPK signaling through ROS-mediated JNK activation. Our findings reveal a rigosertib-dependent mechanism for NLRP3 activation that might be the cause of rigosertib synergistic effect with immune checkpoint blockers to induce cell death in advanced KRAS-mutated non-small cell lung cancer patients. This MOA suggests rigosertib can modulate the mutated RAS pathway no matter the mutation present, now confirmed with clinical data. Further clinical trials in a number of RAS mutated cancers are continuing. Citation Format: Petros Kechagioglou, Camille Dupont, Hajime Yurugi, Alexey Chernobrovkin, Kristina Riegel, Stephen Cosenza, Steven M. Fruchtman, Krishnaraj Rajalingam. CETSA profiling unveils novel targets engaged by anti-tumor drug rigosertib to inhibit RAS-MAPK signaling and trigger NLRP3 inflammasome activation [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A020.

A highly potent small-molecule antagonist of exportin-1 selectively eliminates CD44+CD24- enriched breast cancer stem-like cells.

Breast cancer stem-like cells (BCSCs) have been suggested as the underlying cause of tumor recurrence, metastasis and drug resistance in triple-negative breast cancer (TNBC). Here, we report the discovery and biological evaluation of a highly potent small-molecule antagonist of exportin-1, LFS-1107. We ascertained that exportin-1 (also named as CRM1) is a main cellular target of LFS-1107 by nuclear export functional assay, bio-layer interferometry binding assay and C528S mutant cell line. We found that LFS-1107 significantly inhibited TNBC tumor cells at low-range nanomolar concentration and LFS-1107 can selectively eliminate CD44+CD24- enriched BCSCs. We demonstrated that LFS-1107 can induce the nuclear retention of Survivin and consequent strong suppression of STAT3 transactivation abilities and the expression of downstream stemness regulators. Administration of LFS-1107 can strongly inhibit tumor growth in mouse xenograft model and eradicate BCSCs in residual tumor tissues. Moreover, LFS-1107 can significantly ablate the patient-derived tumor organoids (PDTOs) of TNBC as compared to a few approved cancer drugs. Lastly, we revealed that LFS-1107 can enhance the killing effects of chemotherapy drugs and downregulate multidrug resistance related protein targets. These new findings provide preclinical evidence of defining LFS-1107 as a promising therapeutic agent to deplete BCSCs for the treatment of TNBC.

Is Infantile Hemangioma a Neuroendocrine Tumor?

Infantile hemangioma (IH) is the most common infantile tumor, affecting 5–10% of newborns. Propranolol, a nonselective β-adrenergic receptor (ADRB) antagonist, is currently the first-line treatment for severe IH; however, both its mechanism of action and its main cellular target remain poorly understood. Since betablockers can antagonize the effect of natural ADRB agonists, we postulated that the catecholamine produced in situ in IH may have a role in the propranolol response. By quantifying catecholamines in the IH tissues, we found a higher amount of noradrenaline (NA) in untreated proliferative IHs than in involuted IHs or propranolol-treated IHs. We further found that the first three enzymes of the catecholamine biosynthesis pathway are expressed by IH cells and that their levels are reduced in propranolol-treated tumors. To study the role of NA in the pathophysiology of IH and its response to propranolol, we performed an in vitro angiogenesis assay in which IH-derived endothelial cells, pericytes and/or telocytes were incorporated. The results showed that the total tube formation is sensitive to propranolol only when exogenous NA is added in the three-cell model. We conclude that the IH’s sensitivity to propranolol depends on crosstalk between the endothelial cells, pericytes and telocytes in the context of a high local amount of local NA.

Inhibition of SARS-CoV-2 replication in the lung with siRNA/VIPER polyplexes.

SARS-CoV-2 has been the cause of a global pandemic since 2019 and remains a medical urgency. siRNA-based therapies are a promising strategy to fight viral infections. By targeting a specific region of the viral genome, siRNAs can efficiently downregulate viral replication and suppress viral infection. However, to achieve the desired therapeutic activity, siRNA requires a suitable delivery system. The VIPER (virus-inspired polymer for endosomal release) block copolymer has been reported as promising delivery system for both plasmid DNA and siRNA in the past years. It is composed of a hydrophilic block for condensation of nucleic acids as well as a hydrophobic, pH-sensitive block that, at acidic pH, exposes the membrane lytic peptide melittin, which enhances endosomal escape. In this study, we aimed at developing a formulation for pulmonary administration of siRNA to suppress SARS-CoV-2 replication in lung epithelial cells. After characterizing siRNA/VIPER polyplexes, the activity and safety profile were confirmed in a lung epithelial cell line. To further investigate the activity of the polyplexes in a more sophisticated cell culture system, an air-liquid interface (ALI) culture was established. siRNA/VIPER polyplexes reached the cell monolayer and penetrated through the mucus layer secreted by the cells. Additionally, the activity against wild-type SARS-CoV-2 in the ALI model was confirmed by qRT-PCR. To investigate translatability of our findings, the activity against SARS-CoV-2 was tested ex vivo in human lung explants. Here, siRNA/VIPER polyplexes efficiently inhibited SARS-CoV-2 replication. Finally, we verified the delivery of siRNA/VIPER polyplexes to lung epithelial cells in vivo, which represent the main cellular target of viral infection in the lung. In conclusion, siRNA/VIPER polyplexes efficiently delivered siRNA to lung epithelial cells and mediated robust downregulation of viral replication both in vitro and ex vivo without toxic or immunogenic side effects in vivo, demonstrating the potential of local siRNA delivery as a promising antiviral therapy in the lung.

Damage to the Vascular Endothelium as a Leading Mechanism of COVID-19 Systemic Pathology

An analysis of the molecular and pathophysiological mechanisms of COVID-19 is presented. The endothelium of blood vessels, a kind of “endocrine tree” of the lungs and other organs in which important pathophysiological processes are concentrated, is considered as the target of the aggressive effects of the SARS-CoV-2 coronavirus. Since the main cellular target of viral aggression is the ACE2 enzyme, consideration of its role is the main line of discussion. Coronavirus blocks the activity of ACE2, a natural producer of angiotensins peptides, thus disrupting the balance of hemovascular control. Under normal conditions this mission is performed by the ACE/ACE2 complex, enzymes that control the synthesis and physiological activity of angiotensins and bradykinin peptides. Changes in the ACE/ACE2 axis ratios and cytokine stress are associated with endothelial dysfunction and a number of vascular disorders. The STORM-2 concept is proposed for the first time. According to the concept, the cause of severe organ pathology is the violation of hemostasis, transcellular diffusion, and maintenance of blood pressure.

The SARS-CoV-2 Spike protein alters human cardiac pericyte function and interaction with endothelial cells through a non-infective mechanism involving activation of CD147 receptor signalling

BackgroundHuman cardiac pericytes (PC) were proposed as the main cellular target for SARS-CoV-2 in the heart due to high transcriptional levels of the angiotensin-converting enzyme 2 (ACE2) receptor. Emerging reports indicate CD147/Basigin (BSG), highly expressed in endothelial cells (EC), is an alternative SARS-CoV-2 receptor. To date, the mechanism by which the virus infects and disrupts the heart vascular cells was not identified yet. Moreover, cleaved Spike (S) protein molecules could be released into the bloodstream from the leaking pulmonary epithelial-endothelial barrier in patients with severe COVID-19, opening to the possibility of non-infective diseases in organs distant from the primary site of infection.Purposes(1) to confirm that human primary cardiac PC express ACE2 and CD147; (2) to verify if PC are permissible to SARS-CoV-2 infection; (3) to investigate if the recombinant SARS-CoV-2 S protein alone, without the other viral elements, can trigger molecular signalling and induce functional alterations in PC; (4) to explore which viral receptor is responsible for the observed events.Methods and resultsCardiac PC express both the ACE2 and CD147 receptors at mRNA and protein level. Incubation of PC for up to 5 days with SARS-CoV-2 expressing the green fluorescent protein (GFP) did not show any evidence of cell infection or viral replication. Next, we exposed the PC to the recombinant S protein (5.8 nM) and confirmed that the protein engaged with cellular receptors (western blot analysis of S protein in treated and control PC). Incubation with the S protein increased PC migration (wound closure assay, P<0.01 vs ctrl) and reduced the formation of tubular structures between PC and EC in a Matrigel assay (P<0.01 vs ctrl). Moreover, the S protein promoted the production of pro-inflammatory factors typical of the cytokine storm in PC (ELISA measurement of MCP1, IL-6, IL-1β, TNFα, P<0.05 vs ctrl), and induced the secretion of pro-apoptotic factors responsible for EC death (Caspase 3/7 assay, P<0.05 vs ctrl). Signalling studies revealed that the S protein triggers the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in cardiac PC. The neutralization of CD147, using a blocking antibody, prevented ERK1/2 activation in PC, and was reflected into a partial rescue of the cell functional behaviour (migration and pro-angiogenic capacity). In contrast, blockage of CD147 failed to prevent the pro-inflammatory response in PC.ConclusionsWe propose the novel hypothesis that COVID-19 associated heart's microvascular dysfunction is prompted by circulating S protein molecules rather than by the direct coronavirus infection of PC. Besides, we propose CD147, and not ACE2, as the leading receptor mediating S protein signalling in cardiac PC.FUNDunding AcknowledgementType of funding sources: Foundation. Main funding source(s): BHF project grant “Targeting the SARS-CoV-2 S-protein binding to the ACE2 receptor to preserve human cardiac pericytes function in COVID-19” BHF Centre for Vascular Regeneration II

Direct derivation of human alveolospheres for SARS-CoV-2 infection modeling and drug screening.

Although the main cellular target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is thought to be alveolar cells, the absence of their tractable culture system precludes the development of a clinically relevant SARS-CoV-2 infection model. Here, we establish an efficient human alveolosphere culture method and sphere-based drug testing platform for SARS-CoV-2. Alveolospheres exhibit indolent growth in a Wnt- and R-spondin-dependent manner. Gene expression, immunofluorescence, and electron microscopy analyses reveal the presence of alveolar cells in alveolospheres. Alveolospheres express ACE2 and allow SARS-CoV-2 to propagate nearly 100,000-fold in 3 days of infection. Whereas lopinavir and nelfinavir, protease inhibitors used for the treatment of human immunodeficiency virus (HIV) infection, have a modest anti-viral effect on SARS-CoV-2, remdesivir, a nucleotide prodrug, shows an anti-viral effect at the concentration comparable with the circulating drug level. These results demonstrate the validity of the alveolosphere culture system for the development of therapeutic agents to combat SARS-CoV-2.

Feline Immunodeficiency Virus: characteristics and role in pathology

The review describes structure and biology feline immunodeficiency virus, epidemiology, clinical manifistation, immunological and immunogenetic characteristics of the pathogenesis, principles of diagnosis, treatment and prophylactic. This is a brief overview of the current state of knowledge of this virus. The feline immunodefficiency virus (FIV) is a retrovirus of the Lentivirus genus (Family Retroviridae) was initially isolated from colony of domestic cats in California (USA) in 1986 and has now been recognized as a common feline pathogen worldwide. FIV closely related to HIV, which infect members of Felidae family and it is an importmant viral pathogen worldwide in the domestic cats. FIV these reasons has been studied widely as both an important veterinary pathogen and an animal model for HIV/AIDS. However, it is important to emphasise that humans are not susceptible to FIV infection. The main cellular target for FIV is the CD4+ T cell. FIV causes an immune system disease in domestic cats involving depletion of the CD4+ population of T lymphocytes, increased susceptibility to opportunistic infections, and sometimes death. Seven genetically distinct subtypes has been defined (A,B,C,D,E,F,U-Nzenv).The seroprevalence of feline immunodeficiency virus infection of cats varies markedly between geographic regions. Transmission of FIV is principally by parenteral inoculation of the virus in blood and saliva, presuamably via biting during fighting. Most clinical signs are not directly caused by FIV, clinical signs will be the result of a secondary infection. The virus itself is responsible for immunodeficiency or immune stimulation. Chronic gingivostomatitis one of the most common presenting signs in FIV-infected cats. Methods of diagnosis are included virus isolation (not used routinely), polymerase chain reaction with sensitive and specificities ranging from 40-100%. These techniques result in relatively high numbers of false-positive and false-negative results. Routinely, FIV-infection is diagnosed by detecting antibodies using ELISA and immunochro-matography methods. Western blot analysis is considered the "gold standart" for FIV serology to confirm questionable results. The most common drugs used for treatment of FIV-infection: reverse transcriptase inhibitors drugs, that ingibit firal ensymes, such as DNA or RNA polymerases, integrase ingibitors, protease ingibitors; and interferons. Development of an effective vaccine against FIV is difficult because of the high number and variations of the virus strains. Vaccines that only protect against a single virus variant, have already demonstrated a good efficacy against homologous FIV strains. This review summaries pertinent findings about FIV from work published in a variety research journals.

Neurotoxic potential of reactive astrocytes in canine distemper demyelinating leukoencephalitis

Canine distemper virus (CDV) causes a fatal demyelinating leukoencephalitis in young dogs resembling human multiple sclerosis. Astrocytes are the main cellular target of CDV and undergo reactive changes already in pre-demyelinating brain lesions. Based on their broad range of beneficial and detrimental effects in the injured brain reactive astrogliosis is in need of intensive investigation. The aim of the study was to characterize astrocyte plasticity during the course of CDV-induced demyelinating leukoencephalitis by the aid of immunohistochemistry, immunofluorescence and gene expression analysis. Immunohistochemistry revealed the presence of reactive glial fibrillary acidic protein (GFAP)+ astrocytes with increased survivin and reduced aquaporin 4, and glutamine synthetase protein levels, indicating disturbed blood brain barrier function, glutamate homeostasis and astrocyte maladaptation, respectively. Gene expression analysis revealed 81 differentially expressed astrocyte-related genes with a dominance of genes associated with neurotoxic A1-polarized astrocytes. Accordingly, acyl-coA synthetase long-chain family member 5+/GFAP+, and serglycin+/GFAP+ cells, characteristic of A1-astrocytes, were found in demyelinating lesions by immunofluorescence. In addition, gene expression revealed a dysregulation of astrocytic function including disturbed glutamate homeostasis and altered immune function. Observed findings indicate an astrocyte polarization towards a neurotoxic phenotype likely contributing to lesion initiation and progression in canine distemper leukoencephalitis.

Escherichia coli as a Model for the Description of the Antimicrobial Mechanism of a Cationic Polymer Surface: Cellular Target and Bacterial Contrast Response.

In this study, we use Escherichia coli as a model to investigate the antimicrobial mechanism of a film made of a copolymer based on monomethylether poly(ethylene glycol), methyl methacrylate, and 2-dimethyl(aminoethyl) methacrylate, whose surface is active towards Gram-negative and Gram-positive bacteria. The polymer contains not quaternized amino groups that can generate a charged surface by protonation when in contact with water. For this purpose, we adopted a dual strategy based on the analysis of cell damage caused by contact with the polymer surface and on the evaluation of the cell response to the surface toxic action. The lithic effect on the protoplasts of E. coli showed that the polymer surface can affect the structure of cytoplasmic membranes, while assays of calcein leakage from large unilamellar vesicles at different phospholipid compositions indicated that action on membranes does not need a functionally active cell. On the other hand, the significant increase in sensitivity to actinomycin D demonstrates that the polymer interferes also with the structure of the outer membrane, modifying its permeability. The study on gene expression, based on the analysis of the transcripts in a temporal window where the contact with the polymer is not lethal and the damage is reversible, showed that some key genes of the synthesis and maintenance of the outer membrane structure ( fabR, fadR, fabA, waaA, waaC, kdsA, pldA, and pagP), as well as regulators of cellular response to oxidative stress ( soxS), are more expressed when bacteria are exposed to the polymer surface. All together these results identified the outer membrane as the main cellular target of the antimicrobial surface and indicated a specific cellular response to damage, providing more information on the antimicrobial mechanism. In this perspective, data reported here could play a pivotal role in a microbial growth control strategy based not only on the structural improvements of the materials but also on the possibility of intervening on the cellular pathways involved in the contrast reaction to these and other polymers with similar mechanisms.

Po-323 In vivo monitoring of tumour responses to chemotherapy using multiparameter fluorescence imaging

IntroductionAn increasing number of recent studies suggest that tumour response to chemotherapy cannot be fully described in terms of only interaction of the drug with the main cellular target (e.g. DNA or microtubules) but can include multiple physiological and physicochemical changes. Insight into drug-induced functional alterations in cancer cells and tissues is crucially important for understanding of mechanisms of a drug action and for development new approaches to enable monitoring of the early-treatment response.Material and methodsIn our studies, we focus on multiparameteric analysis of tumour responses to chemotherapy using advanced fluorescence imaging techniques. Previously, we developed methodologies for in vivo probing several parameters, including cytosolic pH, metabolic status, and viscosity of plasma membrane, in mouse tumour models. Mapping of cytosolic pH in tumours is performed using ratiometric genetically encoded sensor SypHer2 and fluorescence whole-body imaging [Shirmanova et al. BBA-GS 2015]. Imaging of cellular metabolism is based on the visualisation of fluorescence intensities and lifetimes of intrinsic metabolic cofactors NAD(P)H and FAD [Shirmanova et al. Sci.Rep. 2017]. Viscosity is measured using molecular BODIPY-based rotor and fluorescence lifetime imaging microscopy (FLIM) [Shimolina et al. Sci.Rep. 2017].Results and discussionsWe showed that acidification of cytosolic pH occurs after therapy with cisplatin in vivo, and this, likely, favours metabolic reorganisation of cells. Treated tumours exhibited a decreased relative contribution from free (cytosolic, protein-unbound) NAD(P)H, indicating a metabolic shift from glycolysis towards oxidative metabolism. It is interesting that optical metabolic imaging could allow early detection of tumour response to chemotherapy, before there are changes in tumour size, and metabolic changes were the same for therapeutic agents with different mechanisms of action (cisplatin, paclitaxel). Moreover, tumours treated with cisplatin displayed decreased plasma membrane viscosity. Presumably, this alteration of viscosity participates in cisplatin-induced apoptosis. Similar changes of pH, energy metabolism and viscosity were previously reported for cisplatin-treated cultured cancer cells.ConclusionThese results, therefore, suggest that all investigated parameters play a role in the cytotoxicity of the drugs and may provide a useful approach for monitoring tumour responses to chemotherapy. The study was supported by the Russian Science Foundation (#14-25-00129).

An in vitro transepithelial migration assay to evaluate the role of neutrophils in Respiratory Syncytial Virus (RSV) induced epithelial damage

Large numbers of neutrophils migrate into the lungs of children with severe Respiratory Syncytial Virus (RSV) disease. It is unclear how these cells contribute to viral clearance and recovery from infection or whether they contribute to disease pathology. We have developed a novel in vitro model to study neutrophil migration through airway epithelial cells (AECs), the main cellular target of RSV infection. Our model reproduces a physiologically relevant cell polarity and directionality of neutrophil migration. Using this model, we found that RSV infected AECs induced rapid neutrophil transepithelial migration. We also detected increased AEC damage associated with RSV infection, with a further increase in epithelial cells shedding from the Transwell membrane following neutrophil migration. This was not observed in the mock infected controls. Neutrophils that migrated through the RSV infected AECs showed increased cell surface expression of CD11B and MPO compared to neutrophils that had not migrated. In conclusion, our in vitro co-culture assay can be used to identify critical mechanisms that mediate epithelial cell damage and promote inflammation in children with severe RSV disease.

Activated Microglia in the Brain: Mitochondrial and Cell Membrane-Associated Targets for Positron Emission Tomography

The emission tomographic imaging of activated microglia in the brain moves into the focus of neuroscientific research with increasing recognition of contributions of early inflammatory processes to neurodegenerative, traumatic, cancerous and infectious diseases of the brain. Whereas the mitochondrial isoform of the 18 kDa translocator protein (TSPO1) has been the main cellular target for positron emission tomography (PET) of this type of cells for decades, alternative marker proteins in the plasma membrane of microglia challenge efforts in ligand development, recently. The present report includes PET approaches using the chemokine receptor CX3CR1 and the FR2 folate receptor in parallel to small molecule PET tracers available for in vivo visualization of the “classical” target TSPO1. It compares first and second generation of TSPO1 ligands as well as new compounds like the tetrahydrocarbazole [18F]GE-180 and the quinazoline [11C]ER176 presumed to reduce polymorphism-related inter-subject variations, with allosteric ligands for the chemokine receptor CX3CR1 and with radio labelled folate conjugates targeting the folate “cargo” receptor FR1 and the FR2 receptor characteristic for anti-inflammatory M2 microglia.

Epigenetic changes in histone acetylation underpin resistance to the topoisomerase I inhibitor irinotecan.

The topoisomerase I (TOP1) inhibitor irinotecan triggers cell death by trapping TOP1 on DNA, generating cytotoxic protein-linked DNA breaks (PDBs). Despite its wide application in a variety of solid tumors, the mechanisms of cancer cell resistance to irinotecan remains poorly understood. Here, we generated colorectal cancer (CRC) cell models for irinotecan resistance and report that resistance is neither due to downregulation of the main cellular target of irinotecan TOP1 nor upregulation of the key TOP1 PDB repair factor TDP1. Instead, the faster repair of PDBs underlies resistance, which is associated with perturbed histone H4K16 acetylation. Subsequent treatment of irinotecan-resistant, but not parental, CRC cells with histone deacetylase (HDAC) inhibitors can effectively overcome resistance. Immunohistochemical analyses of CRC tissues further corroborate the importance of histone H4K16 acetylation in CRC. Finally, the resistant clones exhibit cross-resistance with oxaliplatin but not with ionising radiation or 5-fluoruracil, suggesting that the latter two could be employed following loss of irinotecan response. These findings identify perturbed chromatin acetylation in irinotecan resistance and establish HDAC inhibitors as potential therapeutic means to overcome resistance.

Structural, conformational and thermodynamic aspects of groove-directed-intercalation of flavopiridol into DNA

Certain plant-derived alkaloids and flavonoids have shown propitious cytotoxic acitvity against different types of cancer, having deoxyribose nucleic acid (DNA) as their main cellular target. Flavopiridol, a semi-synthetic derivative of rohitukine (a natural compound isolated from Dysoxylum binectariferum plant), has attained much attention owing to its anticancer potential against various haematological malignancies and solid tumours. This work focuses on investigating interaction between flavopiridol and DNA at molecular level in order to decipher its underlying mechanism of action, which is not well understood. To define direct influence of flavopiridol on the structural, conformational and thermodynamic aspects of DNA, various spectroscopic and calorimetric techniques have been used. ATR-FTIR and SERS spectral outcomes indicate a novel insight into groove-directed-intercalation of flavopiridol into DNA via direct binding with nitrogenous bases guanine (C6=O6) and thymine (C2=O2) in DNA groove together with slight external binding to its sugar–phosphate backbone. Circular dichroism spectral analysis of flavopiridol–DNA complexes suggests perturbation in native B-conformation of DNA and its transition into C-form, which may be localized up to a few base pairs of DNA. UV–visible spectroscopic results illustrate dual binding mode of flavopiridol when interacts with DNA having association constant, Ka = 1.18 × 104 M−1. This suggests moderate type of interaction between flavopiridol and DNA. Further, UV melting analysis also supports spectroscopic outcomes. Thermodynamically, flavopiridol–DNA complexation is an enthalpy-driven exothermic process. These conclusions drawn from this study could be helpful in unveiling mechanism of cytoxicity induced by flavopiridol that can be further applied in the development of flavonoid-based new chemotherapeutics with more specificity and better efficacy.

Oxaliplatin in the era of personalized medicine: from mechanistic studies to clinical efficacy.

Oxaliplatin is a third-generation platinum compound approved for clinical use relatively recently as compared to other drugs of the same class. Its main cellular target is DNA, where similarly to cisplatin and carboplatin it forms cross-links. However, due to a unique indication for colorectal cancer, synergistic interaction with fluoropyrimidines and peculiar toxicity profile, oxaliplatin is different from those compounds. Multiple lines of evidence indicate differences in transport and metabolism, consequences of DNA platination, as well as DNA repair and transduction of DNA damage. Here, we explore the preclinical features that may explain the unique properties of oxaliplatin in the clinics. Among them, the capability to accumulate in tumor cells via organic cation transporters, to kill KRAS mutant cells and to activate immunogenic cell death appears helpful to explain in part its clinical behavior. The continuous investigation of the molecular pharmacology of oxaliplatin is expected to provide clues to the definitions of predictors of drug activity and toxicity to translate to the clinical setting.

African swine fever virus infects macrophages, the natural host cells, via clathrin- and cholesterol-dependent endocytosis

The main cellular target for African swine fever virus (ASFV) is the porcine macrophage. However, existing data about the early phases of infection were previously characterized in non-leukocyte cells such as Vero cells. Here, we report that ASFV enters the natural host cell using dynamin-dependent and clathrin-mediated endocytosis. This pathway is strongly pH-dependent during the first steps of infection in porcine macrophages. We investigated the effect of drugs inhibiting several endocytic pathways in macrophages and compared ASFV with vaccinia virus (VV), which apparently involves different entry pathways. The presence of cholesterol in cellular membranes was found to be essential for a productive ASFV infection while actin-dependent endocytosis and the participation of phosphoinositide-3-kinase (PI3K) activity were other cellular factors required in the process of viral entry. These findings improved our understanding of the ASFV interactions with macrophages that allow for successful viral replication.