Coral-associated bacterial extracts inhibit cellular NF-κB pathway

Marine natural products have recently been recognized as a promising source of NF-κB inhibitors. In this study, extracts of bacterial isolates from the coral Favis sp. were screened for NF-κB modulatory capability using an NF-κB luciferase reporter gene assay. The bacterial extracts had variable effects on NF-κB activity: from the 39 extracts tested, only one exhibited significant NF-κB inhibition and two caused NF-κB up regulation. In addition, we showed that coral bacteria New-33 extract inhibits NF-κB alternative pathway subunits in a non-cytotoxic manner. HPLC analysis confirmed the presence of a low molecular mass compound and NCBI megaBLAST revealed that the New-33 16S rRNA gene sequences are similar to Vibrio mediterranei. This specific modulation of the NF-κB by alternative pathway could contribute to therapeutic implications and also raises questions about host-symbiont interaction in the marine environment

Obstructive Sleep Apnea Syndrome In Vitro Model: Controlled Intermittent Hypoxia Stimulation of Human Stem Cells-Derived Cardiomyocytes

Cardiovascular morbidity is the leading cause of death of obstructive sleep apnea (OSA) syndrome patients. Nocturnal airway obstruction is associated with intermittent hypoxia (IH). In our previous work with cell lines, incubation with sera from OSA patients induced changes in cell morphology, NF-κB activation and decreased viability. A decrease in beating rate, contraction amplitude and a reduction in intracellular calcium signaling was also observed in human cardiomyocytes differentiated from human embryonic stem cells (hESC-CMs). We expanded these observations using a new controlled IH in vitro system on beating hESC-CMs. The Oxy-Cycler system was programed to generate IH cycles. Following IH, we detected the activation of Hif-1α as an indicator of hypoxia and nuclear NF-κB p65 and p50 subunits, representing pro-inflammatory activity. We also detected the secretion of inflammatory cytokines, such as MIF, PAI-1, MCP-1 and CXCL1, and demonstrated a decrease in beating rate of hESC-CMs following IH. IH induces the co-activation of inflammatory features together with cardiomyocyte alterations which are consistent with myocardial damage in OSA. This study provides an innovative approach for in vitro studies of OSA cardiovascular morbidity and supports the search for new pharmacological agents and molecular targets to improve diagnosis and treatment of patients

Secreted Soluble Factors from Tumor-Activated Mesenchymal Stromal Cells Confer Platinum Chemoresistance to Ovarian Cancer Cells

Ovarian cancer (OC) ranks as the second most common type of gynecological malignancy, has poor survival rates, and is frequently diagnosed at an advanced stage. Platinum-based chemotherapy, such as carboplatin, represents the standard-of-care for OC. However, toxicity and acquired resistance to therapy have proven challenging for the treatment of patients. Chemoresistance, a principal obstacle to durable response in OC patients, is attributed to alterations within the cancer cells, and it can also be mediated by the tumor microenvironment (TME). In this study, we report that conditioned medium (CM) derived from murine and human stromal cells, MS-5 and HS-5, respectively, and tumor-activated HS-5, was active in conferring platinum chemoresistance to OC cells. Moreover, CM derived from differentiated murine pre-adipocyte (3T3-L1), but not undifferentiated pre-adipocyte cells, confers platinum chemoresistance to OC cells. Interestingly, CM derived from tumor-activated HS-5 was more effective in conferring chemoresistance than was CM derived from HS-5 cells. Various OC cells exhibit variable sensitivity to CM activity. Exploring CM content revealed the enrichment of a number of soluble factors in the tumor-activated HS-5, such as soluble uPAR (SuPAR), IL-6, and hepatocyte growth factor (HGF). FDA-approved JAK inhibitors were mildly effective in restoring platinum sensitivity in two of the three OC cell lines in the presence of CM. Moreover, Crizotinib, an ALK and c-MET inhibitor, in combination with platinum, blocked HGF’s ability to promote platinum resistance and to restore platinum sensitivity to OC cells. Finally, exposure to 2-hydroxyestardiol (2HE2) was effective in restoring platinum sensitivity to OC cells exposed to CM. Our results showed the significance of soluble factors found in TME in promoting platinum chemoresistance and the potential of combination therapy to restore chemosensitivity to OC cells

MiRNA-124 is a link between measles virus persistent infection and cell division of human neuroblastoma cells.

Measles virus (MV) infects a variety of lymphoid and non-lymphoid peripheral organs. However, in rare cases, the virus can persistently infect cells within the central nervous system. Although some of the factors that allow MV to persist are known, the contribution of host cell-encoded microRNAs (miRNA) have not been described. MiRNAs are a class of noncoding RNAs transcribed from genomes of all multicellular organisms and some viruses, which regulate gene expression in a sequence-specific manner. We have studied the contribution of host cell-encoded miRNAs to the establishment of MV persistent infection in human neuroblastoma cells. Persistent MV infection was accompanied by differences in the expression profile and levels of several host cell-encoded microRNAs as compared to uninfected cells. MV persistence infection of a human neuroblastoma cell line (UKF-NB-MV), exhibit high miRNA-124 expression, and reduced expression of cyclin dependent kinase 6 (CDK6), a known target of miRNA-124, resulting in slower cell division but not cell death. By contrast, acute MV infection of UKF-NB cells did not result in increased miRNA-124 levels or CDK6 reduction. Ectopic overexpression of miRNA-124 affected cell viability only in UKF-NB-MV cells, causing cell death; implying that miRNA-124 over expression can sensitize cells to death only in the presence of MV persistent infection. To determine if miRNA-124 directly contributes to the establishment of MV persistence, UKF-NB cells overexpressing miRNA-124 were acutely infected, resulting in establishment of persistently infected colonies. We propose that miRNA-124 triggers a CDK6-dependent decrease in cell proliferation, which facilitates the establishment of MV persistence in neuroblastoma cells. To our knowledge, this is the first report to describe the role of a specific miRNA in MV persistence

Flavonoids Restore Platinum Drug Sensitivity to Ovarian Carcinoma Cells in a Phospho-ERK1/2-Dependent Fashion

Ovarian cancer (OC) is the second most common type of gynecological malignancy; it has poor survival rates and is frequently (>75%) diagnosed at an advanced stage. Platinum-based chemotherapy, with, e.g., carboplatin, is the standard of care for OC, but toxicity and acquired resistance to therapy have proven challenging. Despite advances in OC diagnosis and treatment, approximately 85% of patients will experience relapse, mainly due to chemoresistance. The latter is attributed to alterations in the cancer cells and is also mediated by tumor microenvironment (TME). Recently, we reported the synthesis of a platinum (IV) prodrug that exhibits equal potency toward platinum-sensitive and resistant OC cell lines. Here, we investigated the effect of TME on platinum sensitivity. Co-culture of OC cells with murine or human mesenchymal stem cells (MS-5 and HS-5, respectively) rendered them resistant to chemotherapeutic agents, including platinum, paclitaxel and colchicine. Platinum resistance was also conferred by co-culture with differentiated murine adipocyte progenitor cells. Exposure of OC cells to chemotherapeutic agents resulted in activation of phospho-ERK1/2. Co-culture with MS-5, which conferred drug resistance, was accompanied by blockage of phospho-ERK1/2 activation. The flavonoids fisetin and quercetin were active in restoring ERK phosphorylation, as well as sensitivity to platinum compounds. Exposure of OC cells to cobimetinib—a MEK1 inhibitor that also inhibits extracellular signal-regulated kinase (ERK) phosphorylation—which resulted in reduced sensitivity to the platinum compound. This suggests that ERK activity is involved in mediating the function of flavonoids in restoring platinum sensitivity to OC co-cultured with cellular components of the TME. Our data show the potential of combining flavonoids with standard therapy to restore drug sensitivity to OC cells and overcome TME-mediated platinum drug resistance

Inhibition of Cysteine Proteases by 6,6′-Dihydroxythiobinupharidine (DTBN) from Nuphar lutea

The specificity of inhibition by 6,6′-dihydroxythiobinupharidine (DTBN) on cysteine proteases was demonstrated in this work. There were differences in the extent of inhibition, reflecting active site structural-steric and biochemical differences. Cathepsin S (IC50 = 3.2 μM) was most sensitive to inhibition by DTBN compared to Cathepsin B, L and papain (IC50 = 1359.4, 13.2 and 70.4 μM respectively). DTBN is inactive for the inhibition of Mpro of SARS-CoV-2. Docking simulations suggested a mechanism of interaction that was further supported by the biochemical results. In the docking results, it was shown that the cysteine sulphur of Cathepsin S, L and B was in close proximity to the DTBN thiaspirane ring, potentially forming the necessary conditions for a nucleophilic attack to form a disulfide bond. Covalent docking and molecular dynamic simulations were performed to validate disulfide bond formation and to determine the stability of Cathepsins-DTBN complexes, respectively. The lack of reactivity of DTBN against SARS-CoV-2 Mpro was attributed to a mismatch of the binding conformation of DTBN to the catalytic binding site of Mpro. Thus, gradations in reactivity among the tested Cathepsins may be conducive for a mechanism-based search for derivatives of nupharidine against COVID-19. This could be an alternative strategy to the large-scale screening of electrophilic inhibitors

Quorum Sensing and NF-κB Inhibition of Synthetic Coumaperine Derivatives from Piper nigrum

Bacterial communication, termed Quorum Sensing (QS), is a promising target for virulence attenuation and the treatment of bacterial infections. Infections cause inflammation, a process regulated by a number of cellular factors, including the transcription Nuclear Factor kappa B (NF-κB); this factor is found to be upregulated in many inflammatory diseases, including those induced by bacterial infection. In this study, we tested 32 synthetic derivatives of coumaperine (CP), a known natural compound found in pepper (Piper nigrum), for Quorum Sensing Inhibition (QSI) and NF-κB inhibitory activities. Of the compounds tested, seven were found to have high QSI activity, three inhibited bacterial growth and five inhibited NF-κB. In addition, some of the CP compounds were active in more than one test. For example, compounds CP-286, CP-215 and CP-158 were not cytotoxic, inhibited NF-κB activation and QS but did not show antibacterial activity. CP-154 inhibited QS, decreased NF-κB activation and inhibited bacterial growth. Our results indicate that these synthetic molecules may provide a basis for further development of novel therapeutic agents against bacterial infections

6,6′-Dihydroxythiobinupharidine (DTBN) Purified from Nuphar lutea Leaves Is an Inhibitor of Protein Kinase C Catalytic Activity

Water lily (Nuphar) bioactive extracts have been widely used in traditional medicine owing to their multiple applications against human ailments. Phyto-active Nuphar extracts and their purified and synthetic derivatives have attracted the attention of ethnobotanists and biochemists. Here, we report that 6,6′-dihydroxythiobinupharidine (DTBN), purified from extracts of Nuphar lutea (L.) Sm. leaves, is an effective inhibitor of the kinase activity of members of the protein kinase C (PKC) family using in vitro and in silico approaches. We demonstrate that members of the conventional subfamily of PKCs, PKCα and PKCγ, were more sensitive to DTBN inhibition as compared to novel or atypical PKCs. Molecular docking analysis demonstrated the interaction of DTBN, with the kinase domain of PKCs depicting the best affinity towards conventional PKCs, in accordance with our in vitro kinase activity data. The current study reveals novel targets for DTBN activity, functioning as an inhibitor for PKCs kinase activity. Thus, this and other data indicate that DTBN modulates key cellular signal transduction pathways relevant to disease biology, including cancer