Diverse roles of SARS-CoV-2 Spike and Nucleocapsid proteins in EndMT stimulation through the TGF-ß-MRTF axis inhibited by aspirin.

BACKGROUND: The SARS-CoV-2 virus causes severe COVID-19 in one-fifth of patients. In addition to high mortality, infection may induce respiratory failure and cardiovascular complications associated with inflammation. Acute or prolonged inflammation results in organ fibrosis, the cause of which might be endothelial disorders arising during the endothelial-mesenchymal transition (EndMT). METHODS: HUVECs and HMEC-1 cells were stimulated with SARS-CoV-2 S (Spike) and N (Nucleocapsid) proteins, and EndMT induction was evaluated by studying specific protein markers via Western blotting. Wound healing and tube formation assays were employed to assess the potential of SARS-CoV-2 to stimulate changes in cell behaviour. MRTF nuclear translocation, ROS generation, TLR4 inhibitors, TGF-ß-neutralizing antibodies, and inhibitors of the TGF-ß-dependent pathway were used to investigate the role of the TGF-ß-MRTF signalling axis in SARS-CoV-2-dependent EndMT stimulation. RESULTS: Both viral proteins stimulate myofibroblast trans-differentiation. However, the N protein is more effective at EndMT induction. The TGF-ß-MRTF pathway plays a critical role in this process. The N protein preferentially favours action through TGF-ß2, whose secretion is induced through TLR4-ROS action. TGF-ß2 stimulates MRTF-A and MRTF-B nuclear translocation and strongly regulates EndMT. In contrast, the Spike protein stimulates TGF-ß1 secretion as a result of ACE2 downregulation. TGF-ß1 induces only MRTF-B, which, in turn, weakly regulates EndMT. Furthermore, aspirin, a common nonsteroidal anti-inflammatory drug, might prevent and reverse SARS-CoV-2-dependent EndMT induction through TGF-ß-MRTF pathway deregulation. CONCLUSION: The reported study revealed that SARS-CoV-2 infection induces EndMT. Moreover, it was demonstrated for the first time at the molecular level that the intensity of the EndMT triggered by SARS-CoV-2 infection may vary and depend on the viral protein involved. The N protein acts through TLR4-ROS-TGF-ß2-MRTF-A/B, whereas the S protein acts through ACE2-TGF-ß1-MRTF-B. Furthermore, we identified aspirin as a potential anti-fibrotic drug for treating patients with SARS-CoV-2 infection.

Evening Primrose Extract Modulates TYMS Expression via SP1 Transcription Factor in Malignant Pleural Mesothelioma.

PURPOSE: To determine the mechanism of EPE in downregulating TYMS in MPM cancer. METHODS: The TYMS mRNA expression with epithelial-to-mesenchymal transition biomarkers and nuclear factor SP1 was assessed using the GEO database in a data set of MPM patients (GSE51024). Invasive MPM cell lines were in vitro models for the investigation of TYMS expression after EPE treatment. The tyms promoter SP1 binding sequences were determined using Genomatix v 3.4 software Electrophoretic mobility shift and dual-luciferase reporter assays revealed specific SP1 motifs in the interaction of EPE and reference compounds. Chromatin immunoprecipitation and Re-ChIP were used for the co-occupancy study. RESULTS: In MPM patients, a positive correlation of overexpressed TYMS with mesenchymal TWIST1, FN1 and N-cadherin was observed. EPE and its major components, gallic and ellagic acid (GA and EA, respectively), downregulated TYMS in invasive MPM cells by interacting with particular SP1 motifs on the tyms promoter. The luciferase constructs confirmed the occupation of two SP1 regulatory regions critical for the promotion of TYMS expression. Both EPE and reference standards influenced SP1 translocation into the nucleus. CONCLUSION: EPE components reduced TYMS expression by occupation of SP1 motifs on the tyms promoter and reversed the EMT phenotype of invasive MPM cells. Further in-depth analysis of the molecular docking of polyphenol compounds with SP1 regulatory motifs is required.

SARS-CoV-2 S and N protein peptides drive invasion abilities of colon cancer cells through TGF-ß1 regulation.

The COVID-19 pandemic led to the delay of colorectal cancer (CRC) diagnosis, which causes CRC to be treated at more advanced, often metastatic stages. Unfortunately, there is no effective treatment for metastatic CRC stages, which are considered the leading cause of patients' death. The mortality induced by SARS-CoV-2 is significantly higher in cancer patients than in patients with other diseases. Interestingly, COVID-19 patients often develop fibrosis which depends on epithelial-mesenchymal transition (EMT) - the process also involved in cancer progression. The study aimed to verify whether SARS-CoV-2 induces EMT and consequently increases the invasion potential of colon cancer cells. CRC cells were stimulated with SARS-CoV-2 S and N protein peptides and epithelial and mesenchymal markers were analysed with Western blotting to detect the occurrence of the EMT. The migration, invasion assays and MMP-7 secretion were employed to evaluate the potential of SARS-CoV-2 to stimulate the cells invasion in vitro. ELISA assay, TGF-ß1 neutralizing antibodies, TGF-ßR silencing and inhibitors were used to investigate the role of the TGF-ß1 signalling pathways in the SARS-CoV-2-dependent CRC stimulation. The SARS-CoV-2 induced EMT, which increased the invasion ability of CRC cells. Moreover, the SARS-CoV-2 proteins drive colon cancer cell invasion through TGF-ß1. Additionally, secreted TGF-ß1 induced a bystander effect in colon cancer cells. However, blocking TGF-ß1/Smad- and -non-Smad-dependent pathways suppressed the SARS-CoV-2-induced invasiveness of CRC. In conclusion, we revealed that SARS-CoV-2 stimulates the invasion abilities of CRC by regulating TGF-ß1-induced EMT. Our results provide a theoretical basis for using anti-TGF-ß1 therapy to reduce the risk of CRC metastasis during SARS-CoV-2 infection.

Whole grain metabolite 3,5-dihydroxybenzoic acid is a beneficial nutritional molecule with the feature of a double-edged sword in human health: a critical review and dietary considerations.

Nonprocessed foodstuffs of plant origin, especially whole-grain cereals, are considered to be health-promoting components of the human diet. While most of their well-studied effects derive from their high fiber content and low glycemic index, the presence of underrated phenolic phytonutrients has recently been brought to the attention of nutritionists. In this review, we report and discuss findings on the sources and bioactivities of 3,5-dihydroxybenzoic acid (3,5-DHBA), which is both a direct dietary component (found, e.g., in apples) and, more importantly, a crucial metabolite of whole-grain cereal-derived alkylresorcinols (ARs). 3,5-DHBA is a recently described exogenous agonist of the HCAR1/GPR81 receptor. We concentrate on the HCAR1-mediated effects of 3,5-DHBA in the nervous system, on the maintenance of cell stemness, regulation of carcinogenesis, and response to anticancer therapy. Unexpectedly, malignant tumors take advantage of HCAR1 expression to sense 3,5-DHBA to support their growth. Thus, there is an urgent need to fully identify the role of whole-grain-derived 3,5-DHBA during anticancer therapy and its contribution in the regulation of vital organs of the body via its specific HCAR1 receptor. We discuss here in detail the possible consequences of the modulatory capabilities of 3,5-DHBA in physiological and pathological conditions in humans.

Lactate drives cellular DNA repair capacity: Role of lactate and related short-chain fatty acids in cervical cancer chemoresistance and viral infection.

The characteristic feature of a cancer microenvironment is the presence of a highly elevated concentration of L-lactate in the tumor niche. The lactate-rich environment is also maintained by commensal mucosal microbiota, which has immense potential for affecting cancer cells through its receptoric and epigenetic modes of action. Some of these lactate activities might be associated with the failure of anticancer therapy as a consequence of the drug resistance acquired by cancer cells. Upregulation of cellular DNA repair capacity and enhanced drug efflux are the most important cellular mechanisms that account for ineffective radiotherapy and drug-based therapies. Here, we present the recent scientific knowledge on the role of the HCA1 receptor for lactate and lactate intrinsic activity as an HDAC inhibitor in the development of an anticancer therapy-resistant tumor phenotype, with special focus on cervical cancer cells. In addition, a recent study highlighted the viable role of interactions between mammalian cells and microorganisms in the female reproductive tract and demonstrated an interesting mechanism regulating the efficacy of retroviral transduction through lactate-driven modulation of DNA-PKcs cellular localization. To date, very few studies have focused on the mechanisms of lactate-driven enhancement of DNA repair and upregulation of particular multidrug-resistance proteins in cancer cells with respect to their intracellular regulatory mechanisms triggered by lactate. This review presents the main achievements in the field of lactate impact on cell biology that may promote undesirable alterations in cancer physiology and mitigate retroviral infections.

Evening primrose seed extract rich in polyphenols modulates the invasiveness of colon cancer cells by regulating the TYMS expression.

Natural polyphenols are plant metabolites exhibiting a broad range of biological activities. Among them, anticancer properties seem to be very desirable. This study examined the anticancer and anti-metastatic properties of the polyphenol-rich extract from the evening primrose seeds (EPE). In vitro and in vivo studies performed in colorectal cancer (CRC) cell lines and AOM-DSS-induced colitis-associated colon cancer in mice revealed the EPE anticancer properties. Furthermore, we studied the EPE activity on metastatic abilities and showed that the EPE inhibited invasiveness in the following models (cells isolated from patients with different invasive stages and cells with induced invasion by either Snail overexpression or CAF stimulation). More importantly, we also demonstrated that the EPE decreases the cell invasiveness of 5-fluorouracil (5-FU) resistant CRC cells. The inhibition of metastasis correlated with a decrease in thymidylate synthetase (TYMS), which has recently been associated with metastatic phenotype development. Our results indicate that the EPE might be an effective anticancer agent in suppressing colon cancer metastasis regardless of the invasiveness cause. Based on these findings, we concluded that the used EPE extract rich in polyphenols inhibits cell invasion by TYMS downregulation.

Value choices in European COVID-19 vaccination schedules: how vaccination prioritization differs from other forms of priority setting.

With the limited initial availability of COVID-19 vaccines in the first months of 2021, decision-makers had to determine the order in which different groups were prioritized. Our aim was to find out what normative approaches to the allocation of scarce preventive resources were embedded in the national COVID-19 vaccination schedules. We systematically reviewed and compared prioritization regulations in 27 members of the European Union, the United Kingdom, and Israel. We differentiated between two types of priority categories: groups that have increased infection fatality rate (IFR) compared to the average for the general population and groups chosen because their members experience increased risk of being infected (ROI). Our findings show a clear trend: all researched schedules prioritized criteria referring to IFR (being over 65 years old and coexisting health conditions) over the ROI criteria (eg occupation and housing conditions). This is surprising since, in the context of treatment, it is common and justifiable to adopt different allocation principles (eg introducing a saving more life-year approach or prioritizing younger patients). We discuss how utilitarian, prioritarian, and egalitarian principles can be applied to interpret normative differences between the allocation of curative and preventive interventions.

Thymidylate Synthase Overexpression Drives the Invasive Phenotype in Colon Cancer Cells.

Thymidylate synthase (TYMS) is the crucial enzymatic precursor for DNA biosynthesis and, therefore, the critical target for numerous types of chemotherapy, including the most frequently applied agent in colon cancer treatment 5-fluorouracil (5-FU). TYMS also seems to be associated with cancer metastasis and acquiring mesenchymal character by tumor cells during epithelial-mesenchymal transition (EMT). Based on that knowledge, we decided to investigate the role of TYMS in the modulation of invasive ability in colon cancer cells, where its effect on cancer metastasis has not been studied in detail before. We employed colon cancer cells isolated from different stages of tumor development, cells undergoing EMT, and TYMS overexpressing cells. The elongation ratio, cell migration, invasion assay, and MMP-7 secretion were applied to analyze the cell behavior. Important epithelial and mesenchymal markers characteristic of EMT were examined at the protein level by Western blot assay. Overall, our study showed a correlation between TYMS level and invasion ability in colon cancer cells and, above all, a crucial role of TYMS in the EMT regulation. We postulate that chemotherapeutics that decrease or inhibit TYMS expression could increase the effectiveness of the therapy in patients with colon cancer, especially in the metastatic stage.

Lactate Suppresses Retroviral Transduction in Cervical Epithelial Cells through DNA-PKcs Modulation.

Recently, we have shown the molecular basis for lactate sensing by cervical epithelial cells resulting in enhanced DNA repair processes through DNA-PKcs regulation. Interestingly, DNA-PKcs is indispensable for proper retroviral DNA integration in the cell host genome. According to recent findings, the mucosal epithelium can be efficiently transduced by retroviruses and play a pivotal role in regulating viral release by cervical epithelial cells. This study examined the effects of lactate on lentiviral transduction in cervical cancer cells (HeLa, CaSki, and C33A) and model glioma cell lines (DNA-PKcs proficient and deficient). Our study showed that L- and D-lactate enhanced DNA-PKcs presence in nuclear compartments by between 38 and 63%, which corresponded with decreased lentiviral transduction rates by between 15 and 36%. Changes in DNA-PKcs expression or its inhibition with NU7441 also greatly affected lentiviral transduction efficacy. The stimulation of cells with either HCA1 agonist 3,5-DHBA or HDAC inhibitor sodium butyrate mimicked, in part, the effects of L-lactate. The inhibition of lactate flux by BAY-8002 enhanced DNA-PKcs nuclear localization which translated into diminished lentiviral transduction efficacy. Our study suggests that L- and D-lactate present in the uterine cervix may play a role in the mitigation of viral integration in cervical epithelium and, thus, restrict the viral oncogenic and/or cytopathic potential.

Cytoskeleton Reorganization in EndMT-The Role in Cancer and Fibrotic Diseases.

Chronic inflammation promotes endothelial plasticity, leading to the development of several diseases, including fibrosis and cancer in numerous organs. The basis of those processes is a phenomenon called the endothelial-mesenchymal transition (EndMT), which results in the delamination of tightly connected endothelial cells that acquire a mesenchymal phenotype. EndMT-derived cells, known as the myofibroblasts or cancer-associated fibroblasts (CAFs), are characterized by the loss of cell-cell junctions, loss of endothelial markers, and gain in mesenchymal ones. As a result, the endothelium ceases its primary ability to maintain patent and functional capillaries and induce new blood vessels. At the same time, it acquires the migration and invasion potential typical of mesenchymal cells. The observed modulation of cell shape, increasedcell movement, and invasion abilities are connected with cytoskeleton reorganization. This paper focuses on the review of current knowledge about the molecular pathways involved in the modulation of each cytoskeleton element (microfilaments, microtubule, and intermediate filaments) during EndMT and their role as the potential targets for cancer and fibrosis treatment.

Polyphenol Extract from Evening Primrose (Oenothera paradoxa) Inhibits Invasion Properties of Human Malignant Pleural Mesothelioma Cells.

Extracts from the defatted evening primrose (Oenothera paradoxa Hudziok) seeds are the source of a range of stable polyphenolic compounds, including ellagic acid, gallic acid, and catechin. Our studies evaluate, for the first time, the influence of evening primrose isopropanol extract (EPE) on malignant pleural mesothelioma (MPM) cells. MPM is rarely diagnosed, its high aggressiveness and frequently noted chemoresistance limit its treatment schemes and it is characterized by low prognostic features. Here, we demonstrate that EPE inhibited MPM growth in a dose-dependent manner in cells with increased invasion properties. Moreover, EPE treatment resulted in cell cycle arrest in the G2/M phase and increased apoptosis in invasive MPM cell lines. Additionally, EPE strongly limited invasion and MMP-7 secretion in MPM cancer cells. Our original data provide evidence about the potential anti-invasive effects of EPE in MPM therapy treatment.

The New Model of Snail Expression Regulation: The Role of MRTFs in Fast and Slow Endothelial-Mesenchymal Transition.

Endothelial-mesenchymal transition (EndMT) is a crucial phenomenon in regulating the development of diseases, including cancer metastasis and fibrotic disorders. The primary regulators of disease development are zinc-finger transcription factors belonging to the Snail family. In this study, we characterized the myocardin-related transcription factor (MRTF)-dependent mechanisms of a human snail promoter regulation in TGF-ß-stimulated human endothelial cells. Although in silico analysis revealed that the snail promoter's regulatory fragment contains one GCCG and two SP1 motifs that could be occupied by MRTFs, the genetic study confirmed that MRTF binds only to SP1 sites to promote snail expression. The more accurate studies revealed that MRTF-A binds to both SP1 elements, whereas MRTF-B to only one (SP1near). Although we found that each MRTF alone is capable of inducing snail expression, the direct cooperation of these proteins is required to reinforce snail expression and promote the late stages of EndMT within 48 hours. Furthermore, genetic and biochemical analysis revealed that MRTF-B alone could induce the late stage of EndMT. However, it requires a prolonged time. Therefore, we concluded that MRTFs might cause EndMT in a fast- and slow-dependent manner. Based on MRTF-dependent Snail upregulation, we recognized that TGF-ß1, as an MRTF-B regulator, is involved in slow EndMT induction, whereas TGF-ß2, which altered both MRTF-A and MRTF-B expression, promotes a fast EndMT process.

Endothelial Cells in the Tumor Microenvironment.

Angiogenesis is a critical process required for tumor progression. Newly formed blood vessels provide nutrition and oxygen to the tumor contributing to its growth and development. However, endothelium also plays other functions that promote tumor metastasis. It is involved in intravasation, which allows invasive cancer cells to translocate into the blood vessel lumen. This phenomenon is an important stage for cancer metastasis. Besides direct association with cancer development, endothelial cells are one of the main sources of cancer-associated fibroblasts (CAFs). The heterogeneous group of CAFs is the main inductor of migration and invasion abilities of cancer cells. Therefore, the endothelium is also indirectly responsible for metastasis. Considering the above, the endothelium is one of the important targets of anticancer therapy. In the chapter, we will present mechanisms regulating endothelial function, dependent on cancer and cancer niche cells. We will focus on possibilities of suppressing pro-metastatic endothelial functions, applied in anti-cancer therapies.

Transforming Growth Factor-ß Receptor Internalization via Caveolae Is Regulated by Tubulin-ß2 and Tubulin-ß3 during Endothelial-Mesenchymal Transition.

Fibrotic disorders, which are caused by long-term inflammation, are observed in numerous organs. These disorders are regulated mainly through transforming growth factor (TGF)-ß family proteins by a fundamental cellular mechanism, known as the endothelial-mesenchymal transition. Therefore, there is a pressing need to identify the mechanisms and potential therapeutic targets that enable the inhibition of endothelial transdifferentiation. This study is the first to demonstrate that glycosylation of tubulin-ß2 and tubulin-ß3 in microtubules enhances sensitivity to TGF-ß1 stimulation in human microvascular endothelial cells. We observed that the microtubules enriched in glycosylated tubulin-ß2 and tubulin-ß3 were necessary for caveolae-dependent TGF-ß receptor internalization. Post-translational modulation is critical for the generation of myofibroblasts through endothelial-mesenchymal transition during fibrosis development. We suggest that microtubule glycosylation may become the target of new effective therapies for patients with recognized fibrotic diseases.

TUBB4B Downregulation Is Critical for Increasing Migration of Metastatic Colon Cancer Cells.

Tumor metastasis, the major problem for clinical oncology in colon cancer treatment, is linked with an epithelial-mesenchymal transition (EMT). The observed cellular transformation in this process is manifested by cell elongation, enhanced cell migration and invasion ability, coordinated by cytoskeleton reorganization. In the present study, we examined the role of tubulin-ß4 (TUBB4B) downregulation that occurs during EMT in colon cancer cells, in the modulation of the function of microtubules. Based on biochemical and behavioral analysis (transmigration) we posit that the decrease of the TUBB4B level is critical for microtubule-vimentin interaction and contributes to the maintenance of polarity in migrating cells. The microscopic studies revealed that TUBB4B decrease is accompanied by cell elongation and increased number of matured focal adhesion sites, which is a characteristic of the cell metastatic stage. We also demonstrated faster polymerization of microtubules in cells with a lower level of TUBB4B. Simultaneous TUBB3 upregulation, reported during EMT, acts additively in this process. Our studies suggest that the protein level of TUBB4B could be used as a marker for detection of the preinvasive stages of the colon cancer cells. We also concluded that chemotherapy enriched to increase TUBB4B level and/or to stabilize microtubule polymerization might more effectively prevent metastasis in colon cancer development.

Nonsteroidal Anti-Inflammatory Drugs Prevent Vincristine-Dependent Cancer-Associated Fibroblasts Formation.

Vincristine is used in the clinical treatment of colon cancer, especially in patients diagnosed in the advanced phase of cancer development. Unfortunately, similar to other agents used during antitumor therapy, vincristine might induce chemoresistance. Studies of this process focus mainly on the analysis of the molecular mechanisms within cancer, usually ignoring the role of stromal cells. Our present findings confirm that vincristine stimulates the secretion of tumor growth factors class beta and interleukin-6 from cancer-associated fibroblasts as a result of paracrine stimulation by cancer cells. Based on alterations in morphology, modulation of capillary formation, and changes in endothelial and mesenchymal marker profile, our findings demonstrate that higher levels of tumor growth factor-ßs and interleukin-6 enhance cancer-associated fibroblast-like cell formation through endothelial-mesenchymal transition and that nonsteroidal anti-inflammatory drug treatment (aspirin and ibuprofen) is able to inhibit this phenomenon. The process appears to be regulated by the rate of microtubule polymerization, depending on ß-tubulin composition. While higher levels of tubulin-ß2 and tubulin-ß4 caused slowed polymerization and reduced the level of factors secreted to the extracellular matrix, tubulin-ß3 induced the opposite effect. We conclude that nonsteroidal anti-inflammatory drugs should be considered for use during vincristine monotherapy in the treatment of patients diagnosed with colorectal cancer.

Correction: Colchicine metallocenyl bioconjugates showing high antiproliferative activities against cancer cell lines.

Correction for 'Colchicine metallocenyl bioconjugates showing high antiproliferative activities against cancer cell lines' by Karolina Kowalczyk et al., Dalton Trans., 2017, 46, 17041-17052.

Colchicine metallocenyl bioconjugates showing high antiproliferative activities against cancer cell lines.

A series of ferrocenyl and ruthenocenyl conjugates with colchicine bearing a 1,2,3-triazole moiety were synthesized and their anticancer properties were evaluated. We found that the most potent metallocenyl derivatives Rc4 and Rc5 are 6-7 times more cytotoxic toward HepG2 cells, while Fc4 and Fc5 are two times more cytotoxic toward HCT116 cells as colchicine. We also found that compounds Fc4, Fc5, Rc1 and Rc3-Rc5 are able to induce apoptosis, while compound Fc2 arrests mitosis.

Synthesis and in†vitro Biological Evaluation of Ferrocenyl Side-Chain-Functionalized Paclitaxel Derivatives.

Taxanes, including paclitaxel, are widely used in cancer therapy. In an attempt to overcome some of the disadvantages entailed with taxane chemotherapy, we devised the synthesis of ferrocenyl-functionalized paclitaxel derivatives and studied their biological properties. The cytotoxic activity was measured with a panel of human cancer cell lines of various tissue origin, including multidrug-resistant lines. A structure-activity study of paclitaxel ferrocenylation revealed the N-benzoyl-ferrocenyl-substituted derivative to be the most cytotoxic. In contrast, substitution of the 3'-phenyl group of paclitaxel with a ferrocenyl moiety led to less potent antiproliferative compounds. However, these agents were able to overcome multidrug resistance, as they were virtually unrecognized by ABCB1, a major cellular exporter of taxanes. Interestingly, the redox properties of these ferrocenyl derivatives appear to play a less important role in their mode of action, as there was no correlation between intracellular redox activity and cytotoxicity/cell-cycle distribution. The antiproliferative activity of ferrocenyl taxanes strongly depends on the substitution position, and good tubulin polymerization inducers, as confirmed by molecular docking, were usually more cytotoxic, whereas compounds with stronger pro-oxidative properties exhibited lower antiproliferative activity.