Assessing the Presence of Hematopoietic Stem and Progenitor Cells in Mouse Spleen.

Transplantation of hematopoietic material into recipient mice is an assay routinely used to determine the presence and function of hematopoietic stem and progenitor cells (HSPCs) in vivo . The principle of the method is to transplant donor cells being tested for HSPCs into a recipient mouse following bone marrow ablation and testing for reconstitution of hematopoiesis. Congenic mouse strains where donor and recipient differ by a distinct cell surface antigen (commonly CD45.1 versus CD45.2) are used to distinguish between cells derived from the donor and any residual recipient cells. Typically, the transplantation is performed using bone marrow cells, which are enriched for HSPCs. Here, we describe an analogous procedure using hematopoietic material from spleen, allowing detection of functional progenitors and/or stem cells in the spleen that can occur under certain pathologies. Key to the success of this procedure is the prior removal of mature T cells from the donor sample, to minimize graft versus host reactions. As such, this protocol is highly analogous to standard bone marrow transplant procedures, differing mainly only in the source of stem cells (spleen rather than bone marrow) and the recommendation for T cell depletion to avoid potential immune incompatibilities. Graphical abstract: Schematic overview for assessment of stem cells in spleen by transplantation. Single cell suspensions from spleens are depleted of potentially pathogenic mature T lymphocytes by magnetic bead immunoselection using biotinylated antibodies against CD4 and CD8, followed by streptavidin magnetic beads, which are subsequently removed by using a magnet (MojoSort, Biolegend). Successful T cell depletion is then evaluated by Fluorescence Activated Cell Sorting (FACS). T-cell depleted cell suspension is injected intravenously through the retro-orbital sinus into lethally irradiated recipients. Recipients are analyzed for successful engraftment by FACS analysis for the presence of donor-derived mature hematopoietic lineages in the peripheral blood. A second serial transplantation can be used to document the presence of long-term reconstituting stem cells in the periphery of the original donor mice.

Tonic interferon restricts pathogenic IL-17-driven inflammatory disease via balancing the microbiome.

Maintenance of immune homeostasis involves a synergistic relationship between the host and the microbiome. Canonical interferon (IFN) signaling controls responses to acute microbial infection, through engagement of the STAT1 transcription factor. However, the contribution of tonic levels of IFN to immune homeostasis in the absence of acute infection remains largely unexplored. We report that STAT1 KO mice spontaneously developed an inflammatory disease marked by myeloid hyperplasia and splenic accumulation of hematopoietic stem cells. Moreover, these animals developed inflammatory bowel disease. Profiling gut bacteria revealed a profound dysbiosis in the absence of tonic IFN signaling, which triggered expansion of TH17 cells and loss of splenic Treg cells. Reduction of bacterial load by antibiotic treatment averted the TH17 bias and blocking IL17 signaling prevented myeloid expansion and splenic stem cell accumulation. Thus, tonic IFNs regulate gut microbial ecology, which is crucial for maintaining physiologic immune homeostasis and preventing inflammation.

Mitochondrial STAT3 regulates antioxidant gene expression through complex I-derived NAD in triple negative breast cancer.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor with roles in inflammation and tumorigenicity. A fraction of STAT3 localizes in mitochondria, where it augments tumorigenesis via regulation of mitochondrial functions, including modulation of respiration and redox status. We show a novel mechanism for mitochondrial STAT3 regulation of redox homeostasis in triple-negative breast cancer cells. Loss of STAT3 diminished complex I dehydrogenase activity and impaired NAD+ regeneration, leading to impaired expression of glutathione biosynthetic genes and other antioxidant genes. Expressing mitochondrially restricted STAT3 or replenishment of the cellular NAD pool restored antioxidant gene expression, as did complementation of the NADH dehydrogenase activity by expression of the STAT3-independent yeast dehydrogenase, NDI1. These NAD-regulated processes contributed to malignant phenotypes by promoting clonal cell growth and migration. Proximity interaction and protein pull-down assays identified three components of complex I that associated with mitochondrial STAT3, providing a potential mechanistic basis for how mitochondrial STAT3 affects complex I activity. Our data document a novel mechanism through which mitochondrial STAT3 indirectly controls antioxidant gene regulation through a retrograde NAD+ signal that is modulated by complex I dehydrogenase activity.

STAT3 Inhibitor OPB-51602 Is Cytotoxic to Tumor Cells Through Inhibition of Complex I and ROS Induction.

STAT3 is a transcription factor involved in several cellular activities including inflammation, proliferation, and survival, but it also plays a non-transcriptional role in modulating mitochondrial metabolism. Given its diverse functions in human cancers, it is an emerging therapeutic target. Here we show that OPB-51602, a small molecule inhibitor of STAT3, is highly toxic in a STAT3-dependent manner. Specifically, drug toxicity depends on mitochondrial STAT3 as tumor cells expressing only a mitochondrially restricted form of STAT3 are sensitive to the compound, whereas STAT3-null cells are protected. OPB-51602 inhibited complex I activity and led to increased ROS production, which in turn induced mitophagy, actin rearrangements, and cell death. Cells undergoing reduced oxidative phosphorylation or expressing NDI1 NADH dehydrogenase from Saccharomyces cerevisiae, which bypasses mammalian complex I, were resistant to OPB-51602 toxicity. These results show that targeting mitochondrial STAT3 function causes synthetic lethality through complex I inhibition that could be exploited for cancer chemotherapy.

Mitochondrial dysfunction induced by a SH2 domain-targeting STAT3 inhibitor leads to metabolic synthetic lethality in cancer cells.

In addition to its canonical role in nuclear transcription, signal transducer and activator of transcription 3 (STAT3) is emerging as an important regulator of mitochondrial function. Here, we demonstrate that a novel inhibitor that binds with high affinity to the STAT3 SH2 domain triggers a complex cascade of events initiated by interference with mitochondrial STAT3 (mSTAT3). The mSTAT3-drug interaction leads to mitochondrial dysfunction, accumulation of proteotoxic STAT3 aggregates, and cell death. The cytotoxic effects depend directly on the drug's ability to interfere with mSTAT3 and mitochondrial function, as demonstrated by site-directed mutagenesis and use of STAT3 knockout and mitochondria-depleted cells. Importantly, the lethal consequences of mSTAT3 inhibition are enhanced by glucose starvation and by increased reliance of cancer cells and tumor-initiating cells on mitochondria, resulting in potent activity in cell cultures and tumor xenografts in mice. These findings can be exploited for eliciting synthetic lethality in metabolically stressed cancer cells using high-affinity STAT3 inhibitors. Thus, this study provides insights on the role of mSTAT3 in cancer cells and a conceptual framework for developing more effective cancer therapies.

Anti-biofilm activity against Staphylococcus aureus MRSA and MSSA of neolignans and extract of Piper regnellii

ABSTRACT Many infections worldwide are associated with bacterial biofilms. The effects of isolated neolignans (conocarpan and eupomathenoid-5) and the dichloromethane extract of Piper regnellii (Miq.) C. DC., Piperaceae, were tested against isolates of methicillin-resistant Staphylococcus aureus and methicillin-sensitive S. aureus biofilms and S. aureus planktonic cells. The dichloromethane extract presented better results than isolated neolignans against all of the biofilms tested, with a minimum inhibitory concentration <400 µg/ml for preformed biofilms and minimal biofilm inhibitory concentration of 15.6 µg/ml for biofilm formation. The minimum inhibitory concentration to planktonic cells was <12.5 µg/ml. These results indicate a good effect of the dichloromethane extract against methicillin-resistant S. aureus and methicillin-sensitive S. aureus biofilms and efficient prophylaxis.

A chemogenomic screening identifies CK2 as a target for pro-senescence therapy in PTEN-deficient tumours.

Enhancement of cellular senescence in tumours triggers a stable cell growth arrest and activation of an antitumour immune response that can be exploited for cancer therapy. Currently, there are only a limited number of targeted therapies that act by increasing senescence in cancers, but the majority of them are not selective and also target healthy cells. Here we developed a chemogenomic screening to identify compounds that enhance senescence in PTEN-deficient cells without affecting normal cells. By using this approach, we identified casein kinase 2 (CK2) as a pro-senescent target. Mechanistically, we show that Pten loss increases CK2 levels by activating STAT3. CK2 upregulation in Pten null tumours affects the stability of Pml, an essential regulator of senescence. However, CK2 inhibition stabilizes Pml levels enhancing senescence in Pten null tumours. Taken together, our screening strategy has identified a novel STAT3-CK2-PML network that can be targeted for pro-senescence therapy for cancer.

Hitting the right spot: Mechanism of action of OPB-31121, a novel and potent inhibitor of the Signal Transducer and Activator of Transcription 3 (STAT3).

STAT3 is a key element in many oncogenic pathways and, like other transcription factors, is an attractive target for development of novel anticancer drugs. However, interfering with STAT3 functions has been a difficult task and very few small molecule inhibitors have made their way to the clinic. OPB-31121, an anticancer compound currently in clinical trials, has been reported to affect STAT3 signaling, although its mechanism of action has not been unequivocally demonstrated. In this study, we used a combined computational and experimental approach to investigate the molecular target and the mode of interaction of OPB-31121 with STAT3. In parallel, similar studies were performed with known STAT3 inhibitors (STAT3i) to validate our approach. Computational docking and molecular dynamics simulation (MDS) showed that OPB-31121 interacted with high affinity with the SH2 domain of STAT3. Interestingly, there was no overlap of the OPB-31121 binding site with those of the other STAT3i. Computational predictions were confirmed by in vitro binding assays and competition experiments along with site-directed mutagenesis of critical residues in the STAT3 SH2 domain. Isothermal titration calorimetry experiments demonstrated the remarkably high affinity of OPB-31121 for STAT3 with Kd (10 nM) 2-3 orders lower than other STAT3i. Notably, a similar ranking of the potency of the compounds was observed in terms of inhibition of STAT3 phosphorylation, cancer cell proliferation and clonogenicity. These results suggest that the high affinity and efficacy of OPB-31121 might be related to the unique features and mode of interaction of OPB-31121 with STAT3. These unique characteristics make OPB-31121 a promising candidate for further development and an interesting lead for designing new, more effective STAT3i.

Phytochemical analysis of Pfaffia glomerata inflorescences by LC-ESI-MS/MS.

Pfaffia glomerata contains high levels of ß-ecdysone, which has shown a range of beneficial pharmacological effects. The present study demonstrated that inflorescences of P. glomerata contain other important bioactive compounds in addition to ß-ecdysone. The identification of compounds from inflorescences using liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was performed for the first time. The eight compounds identified were ß-ecdysone, flavonoid glycosides such as quercetin-3-O-glucoside, kaempferol-3-O-glucoside and kaempferol-3-O-(6-p-coumaroyl)-glucoside, oleanane-type triterpenoid saponins such as ginsenoside Ro and chikusetsusaponin IV, in addition to oleanonic acid and gluconic acid. This study provided information on the phytochemicals contained in P. glomerata inflorescences revealing the potential application of this plant part as raw material for the phytotherapeutic and cosmetic industries.

Antioxidant capacity of the leaf extract obtained from Arrabidaea chica cultivated in Southern Brazil.

Arrabidaea chica leaf extract has been used by people as an anti-inflammatory and astringent agent as well as a remedy for intestinal colic, diarrhea, leucorrhea, anemia, and leukemia. A. chica is known to be a good producer of phenolics. Therefore, in the present study, we investigated its antioxidant activity. The phenolic composition of A. chica leaves was studied by liquid chromatography coupled to diode array detection (LC-DAD) and liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), and isoscutellarein, 6-hydroxyluteolin, hispidulin, scutellarein, luteolin, and apigenin were identified. The extract from leaves of A. chica was tested for antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, ß-carotene bleaching test, and total reactive antioxidant potential (TRAP) method. The crude extract quenched DPPH free radicals in a dose-dependent manner, and the IC50 of the extract was 13.51 µg/mL. The ß-carotene bleaching test showed that the addition of the A. chica extract in different concentrations (200 and 500 µg/mL) prevented the bleaching of ß-carotene at different degrees (51.2% ±3.38% and 94% ±4.61%, respectively). The TRAP test showed dose-dependent correlation between the increasing concentrations of A. chica extract (0.1, 0.5, and 1.0 µg/mL) and the TRAP values obtained by trolox (hydro-soluble vitamin E) 0.4738±0.0466, 1.981±0.1603, and 6.877±1.445 µM, respectively. The 2 main flavonoids, scutellarein and apigenin, were separated, and their antioxidant activity was found to be the same as that of the plant extract. These 2 flavonoids were quantified in the plant extract by using a validated HPLC-UV method. The results of these tests showed that the extract of A. chica had a significant antioxidant activity, which could be attributed to the presence of the mixture of flavonoids in the plant extract, with the main contribution of scutellarein and apigenin.

Modulation of the activity of Sp transcription factors by mithramycin analogues as a new strategy for treatment of metastatic prostate cancer.

Deregulated activity of transcription factors (TFs) of the Sp/KLF family, like Sp1, Sp3 and Sp4, and consequent over-expression of Sp-regulated genes occur frequently in human cancers. This provides the rationale for development of inhibitors of Sp TFs as cancer therapeutics. Mithramycin A (MTM-A) is a natural polyketide that binds GC-rich DNA sequences, inhibits activity of Sp TFs and exhibits potent antitumor activity in experimental systems. However, clinical use of MTM-A is limited by the severe toxicity of the compound. Here, we studied two MTM-A analogues, which had been generated by genetically engineering of the MTM-A biosynthetic pathway, and evaluated their activity in human prostate cancer in cell cultures and mouse models. The compounds, named MTM-SDK and MTM-SK, were highly effective in vitro inhibiting proliferation of prostate cancer cells and transcription of Sp-regulated genes by blocking binding of Sp proteins to the gene promoters. When administered to mice, both compounds were well tolerated with maximum tolerated doses of MTM-SDK and MTM-SK, respectively, 4- and 32- fold higher than MTM-A. After systemic administration, both compounds were cleared rapidly from the bloodstream but maintained plasma levels well above the active concentrations required in vitro for inhibition of Sp TF activity and cell proliferation. Consistently, MTM-SDK and MTM-SK inhibited transcription of Sp-regulated genes in prostate tumor xenografts and exhibited potent antitumor activity in subcutaneous and metastatic tumor xenograft models with no or minimal toxicity. Taken together, these data indicate that MTM-SDK and MTM-SK possess significantly improved pharmacological and toxicological properties compared to MTM-A and represent promising drugs for treatment of advanced prostate cancer.