Vehicle-treated Control Cells Research Articles

Synergistic Activity of Adefovir Dipivoxil and Venetoclax to Target the Bioenergetic Metabolism of Acute Myeloid Leukemia

Adefovir dipivoxil (ADV) is an oral prodrug of the nucleotide analogue adefovir that inhibits viral DNA polymerase and is an FDA-approved drug for the treatment of hepatitis B virus. Preliminary evidence of the antileukemic activity of ADV was recently reported in a patient with acute promyelocytic leukemia (APL; Gong, X., et al. Exp Hematol Oncol, 2022). Herein we report on the antileukemic activity of ADV, alone and in combination with venetoclax (VEN), an oral FDA-approved BCL-2 inhibitor for the treatment of acute myeloid leukemia (AML). Firstly, a significant dose-dependent reduction of cell growth and increase in apoptosis were observed in FLT3-ITD+ MV4-11 (IC50: 97nΜ) and Molm-13 (IC50: 0.816μM) cells, and in primary CD34+ blasts from refractory/relapsed (R/R) AML patients (n=5; IC50: 1.285-3.703μM), exposed to serial concentrations of ADV for 48 hours (hrs). In contrast, no significant changes were observed in ADV-treated CD34+ cells from healthy donors (n=5). Combination of ADV and VEN were synergistic (combination index < 1.0) in reducing cell viability of AML cells (i.e., MV-4-11, U937, KG1 and THP-1) and CD34+ blasts from R/R AML patients (n=3). To gain insights into the molecular mechanisms underlying the antileukemic activity of ADV alone and in combination with VEN, we performed RNA-Seq on AML CD34+ blasts from R/R AML patients (n=6) treated with vehicle, ADV (5 μΜ), VEN (100 nΜ), or ADV+VEN for 24 hrs. Gene set enrichment analysis (GSEA) highlighted upregulation of p53 pathway and apoptosis signaling, and downregulation of oxidative phosphorylation (OXPHOS), glycolysis, and MYC target hallmark gene sets in ADV- and ADV+VEN-treated cells compared with vehicle-treated controls. These results were confirmed by western blot analysis showing a dose-dependent reduction in c-MYC, anti-apoptotic proteins (i.e., BCL-2 and MCL-1), and carnitine palmitoyltransferase-1a [CPT1A, a key enzyme for fatty acid oxidation (FAO), a process that feeds into OXPHOS], and an increase in pro-apoptotic proteins (i.e., BAX and BAK) in ADV- and ADV+VEN-treated MV4-11 and primary AML CD34+ cells, compared with vehicle-treated control cells. Seahorse analysis confirmed a significant reduction of OXPHOS [measured by oxygen consumption rate (OCR)], associated with decreased mitochondrial numbers and sizes evaluated by electron microscopy imaging of AML CD34+ blasts treated with ADV or ADV+VEN compared with vehicle-treated controls. To test in vivo the antileukemic activity of ADV+VEN, we treated a cohort of CD45.1 recipient mice transplanted with BM cells from congenic CD45.2 Mll PTD/WT/Flt3 ITD/ITD AML mice with vehicle, ADV (100mg/kg, oral gavage), VEN (100mg/kg, oral gavage), or ADV+VEN (n=10 per group) for 4 weeks. ADV+VEN-treated mice lived longer (median survival: 60 days after treatment completion) than ADV (20 days, p=0.0001), VEN (20 days, p=0.0001), or vehicle (14 days, p=0.0002) -treated controls (Figure 1). We then performed secondary transplants by engrafting 10 6 BM cells from treated donors into CD45.1 recipient mice (n=10 per group). Recipients of BM from ADV+VEN-treated donors survived longer (median survival: 83 days) than recipients of BM from ADV (60.5 days, p<0.01), VEN (45.5 days, p<0.0001), and vehicle (42.5 days, p<0.0001) -treated donors (Figure 2), confirming a decrease in leukemic stem cell (LSC) burden by ADV+VEN. Next, we examined the antileukemic activity of ADV+VEN in human AML models. We first treated a cohort of NSG mice (n=10 per group) transplanted with luciferase-expressing Molm-13 cells. ADV+VEN-treated mice survived longer (median survival: 59.5 days) than ADV (52 days, p=0.04), VEN (40 days, p<0.0001), and vehicle (40.5 days, p<0.0001) -treated mice. We then treated a cohort of NSG mice (n=8 per group) transplanted with FLT3-ITD+ blasts from a patient with R/R AML. ADV+VEN-treated mice survived longer (median: 38.5 days) than ADV (34.5 days, p=0.035), VEN (25 days, p<0.0001), and vehicle (18.5 days, p<0.0001) -treated mice. In summary, ADV has in vivo anti-leukemic activity in AML likely by interfering with the bioenergetic metabolism of leukemic cells and decreasing their apoptotic threshold. The combination of ADV and VEN is seemingly synergistic and prolonged survival of murine and human AML models, providing a rationale of translating this “all oral” regimen to the clinic for treatment of AML patients, especially those requiring low intensity therapy.

Differential functions of RhoGDIβ in malignant transformation and progression of urothelial cell following N-butyl-N-(4-hydmoxybutyl) nitrosamine exposure

BackgroundFunctional role of Rho GDP-dissociation inhibitor beta (RhoGDIβ) in tumor biology appears to be contradictory across various studies. Thus, the exploration of the molecular mechanisms underlying the differential functions of this protein in urinary bladder carcinogenesis is highly significant in the field. Here, RhoGDIβ expression patterns, biological functions, and mechanisms leading to transformation and progression of human urothelial cells (UROtsa cells) were evaluated following varying lengths of exposure to the bladder carcinogen N-butyl-N-(4-hydmoxybutyl) nitrosamine (BBN).ResultsIt was seen that compared to expression in vehicle-treated control cells, RhoGDIβ protein expression was downregulated after 2-month of BBN exposure, but upregulated after 6-month of exposure. Assessments of cell function showed that RhoGDIβ inhibited UROtsa cell growth in cells with BBN for 2-month exposure, whereas it promoted the invasion of cells treated with BBN for 6 months. Mechanistic studies revealed that 2-month of BBN exposure markedly attenuated DNMT3a abundance, and this led to reduced miR-219a promoter methylation, increased miR-219a binding to the RhoGDIβ mRNA 3’UTR, and reduced RhoGDIβ protein translation. While after 6-mo of BBN treatment, the cells showed increased PP2A/JNK/C-Jun axis phosphorylation and this in turn mediated overall RhoGDIβ mRNA transcription and protein expression as well as invasion.ConclusionsThese findings indicate that RhoGDIβ is likely to inhibit the transformation of human urothelial cells during the early phase of BBN exposure, whereas it promotes invasion of the transformed/progressed urothelial cells in the late stage of BBN exposure. The studies also suggest that RhoGDIβ may be a useful biomarker for evaluating the progression of human bladder cancers.

Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in isolated mouse ventricular myocytes.

The Bruton tyrosine kinase (BTK) inhibitor Ibrutinib is associated with a higher incidence of cardiotoxic side effects including heart failure (HF). Ibrutinib is capable of inhibiting PI3K/Akt signaling in neonatal rat ventricular cardiomyocytes when stimulated with insulin-like growth factor 1 (IGF-1). We therefore hypothesized that Ibrutinib might disrupt IGF-1-mediated activation of intracellular Ca handling in adult mouse cardiomyocytes by inhibiting PI3K/Akt signaling. Isolated ventricular myocytes (C57BL6/J) were exposed to IGF-1 at 10 nmol/L in the presence or absence of Ibrutinib (1 µmol/L) or Acalabrutinib (10 µmol/L; cell culture for 24 ± 2 h). Intracellular Ca handling was measured by epifluorescence (Fura-2 AM) and confocal microscopy (Fluo-4 AM). Ruptured-patch whole-cell voltage-clamp was used to measure ICa. Levels of key cardiac Ca handling proteins were investigated by immunoblots. IGF-1 significantly increased Ca transient amplitudes by ∼83% as compared to vehicle treated control cells. This was associated with unaffected diastolic Ca, enhanced SR Ca loading and increased ICa. Co-treatment with Ibrutinib attenuated both the IGF-1-mediated increase in SR Ca content and in ICa. IGF-1 treated cardiomyocytes had significantly increased levels of pS473Akt/Akt and SERCA2a expression as compared to cells concomitantly treated with IGF-1 and Ibrutinib. SR Ca release (as assessed by Ca spark frequency) was unaffected by either treatment. In order to test for potential off-target effects, second generation BTK inhibitor Acalabrutinib with greater BTK selectivity and lower cardiovascular toxicity was tested for IGF1-mediated activation of intracellular Ca handling. Acalabrutinib induced similar effects on Ca handling in IGF-1 treated cultured myocytes as Ibrutinib in regard to decreased Ca transient amplitude and slowed Ca transient decay, hence implying a functional class effect of BTK inhibitors in cardiac myocytes. Inhibition of BTK by Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in adult ventricular mouse myocytes in the face of disrupted Akt signaling and absent SERCA2a upregulation.

Cholesterol-lowering activity of adzuki bean (Vigna angularis) polyphenols.

Adzuki beans (ABs; Vigna angularis) were reported to show potential for prevention of cholesterol absorption and lowering of the blood cholesterol level. However, the main active compounds and some cellular effects remain unknown. In this study, we evaluated the potential cholesterol-lowering effects of (+)-catechin 7-O-β-D-glucopyranoside (C7G) and (+)-epicatechin 7-O-β-D-glucopyranoside (E7G), identified as abundant polyphenols in ABs. To investigate the cholesterol-lowering activity in vitro, cholesterol micelles, bile acids, and Caco-2 cells as an intestinal model were used in the study. C7G and E7G each inhibited micellar solubility in a dose-dependent manner, and their inhibitory activity was as strong as that of (+)-catechin (IC50 values: C7G, 0.23 ± 0.03mg/ml; E7G, 0.22 ± 0.02mg/ml; (+)-catechin, 0.26 ± 0.11mg/ml). The AB polyphenols showed binding activity toward bile acids and changed them into an insoluble form. When Caco-2 cells were treated with C7G or E7G, the amount of incorporated cholesterol was significantly decreased compared with vehicle-treated control cells, and no cytotoxicity was observed under the experimental conditions used. Meanwhile, quantitative real-time PCR revealed that the mRNA level of the cholesterol transporter NPC1L1 remained unchanged in the treated cells. Taken together, the present findings suggest that C7G and E7G are the main active compounds in ABs, and have the ability to inhibit micellar solubility, bind to bile acids, and suppress cholesterol absorption. The present study supports the health benefits of ABs as a medicinal food and the application of AB polyphenols as medicinal supplements to suppress cholesterol elevation.

ODP651 Abnormal Primary Ciliogenesis in the Developing Mouse Kidney of Glucocorticoid Receptor-Deficient Mice

Abstract Glucocorticoid (GC) hormones have well characterised regulatory roles for the development and maturation of several fetal organs, however, specific developmental roles of GCs in the fetal kidney have not been described (1). We have explored both the expression and localisation of the glucocorticoid receptor (GR) within the developing fetal mouse kidney during organogenesis and the effect on the transcriptome of eliminating GR expression in gene-targeted GR-deficient mice. Staining for the GR by immunohistochemistry at E14.5, E16.5 and E18.5 demonstrated widespread expression of the GR in the renal tubule and the interstitial renal mesenchyme, with strong staining in collecting ducts and increasing expression in mesenchyme up to E18.5. Loss of GR expression in GR-null mice had a profound effect on the renal transcriptome with altered expression (at an FDR of 0.5 and fold change >2. 0) for 454 genes in the GR-null mouse kidney at E18.5, with a greater number of genes showing significantly reduced mRNA levels (305) than increased mRNA levels (99) relative to wild-type controls. Interestingly these deregulated genes included key primary cilia-associated genes, such as centrosomal protein 290 (Cep290), with the largest fold change of -3.6, Cep97, Ccp110, and the primary cilia-associated kinesin Kif3A. In addition, two renal tubule markers, Kidney androgen regulated protein (Kap) (proximal tubule marker) and S100 calcium binding protein G (S100g) (distal tubule marker) were also strongly downregulated with fold changes of -8.61 and -2.5 respectively. Analysis of primary cilia in sections of E18.5 kidney using multicolour confocal microscopy demonstrated that primary cilium length was significantly decreased and abnormally projected from kidney proximal tubule cells in E18.5 GR-null mice (5.23 + 0.12, µm + SEM) compared with wild type controls (6.27+ 0.15, µm + SEM). Finally, in cultured IMCD3 mouse collecting duct cells dexamethasone treatment was shown to increase the length of primary cilia compared to vehicle-treated control cells (vehicle 2.49 + 0. 03, µm + SEM vs dexamethasone 3.17 + 0. 05 µm + SEM), an effect that could be blocked by pre-treatment with the GR antagonist RU486. Taken together these results indicate that GC signalling mediated via the GR contributes either directly or indirectly to the normal formation of primary cilia in the proximal tubule and is therefore required for normal kidney development. This also suggests that abnormalities in GR-regulation of ciliogenesis may underpin or contribute to causes of human ciliopathy conditions. 1. Cole TJ, Short KL, Hooper SB (2019) The Science of Steroids. Sem. Fetal and Neonatal Med. 24: 170-175. Presentation: No date and time listed

Novel histone modifications in microglia derived from a mouse model of chronic pain

As the resident immune cells in the central nervous system, microglia play an important role in the maintenance of its homeostasis. Dysregulation of microglia has been associated with the development and maintenance of chronic pain. However, the relevant molecular pathways remain poorly defined. In this study, we used a mass spectrometry-based proteomic approach to screen potential changes of histone protein modifications in microglia isolated from the brain of control and cisplatin-induced neuropathic pain adult C57BL/6J male mice. We identified several novel microglial histone modifications associated with pain, including statistically significantly decreased histone H3.1 lysine 27 mono-methylation (H3.1K27me1, 54.8% of control) and H3 lysine 56 tri-methylation (7.5% of control), as well as a trend suggesting increased H3 tyrosine 41 nitration. We further investigated the functional role of H3.1K27me1 and found that treatment of cultured microglial cells for 4 consecutive days with 1-10μM of NCDM-64, a potent and selective inhibitor of lysine demethylase 7A, an enzyme responsible for the demethylation of H3K27me1, dose-dependently elevated its levels with a greater than a two-fold increase observed at 10μM compared to vehicle-treated control cells. Moreover, pretreatment of mice with NCDM-64 (10 or 25mg/kg/day, i.p.) prior to cisplatin treatment prevented the development of neuropathic pain in mice. The identification of specific chromatin marks in microglia associated with chronic pain may yield critical insight into the contribution of microglia to the development and maintenance of pain, and opens new avenues for the development of novel nonopioid therapeutics for the effective management of chronic pain.

NRF2 activation protects against valproic acid-induced disruption of neurogenesis in P19 cells

Valproic acid (VPA) is a commonly prescribed antiepileptic drug that causes fetal valproate syndrome (FVS) in developing embryos exposed to it. Symptoms of FVS include neural tube defects (NTDs), musculoskeletal abnormalities, and neurodevelopmental difficulties. One proposed mechanism of VPA-induced developmental toxicity is via oxidative stress, defined as the disruption of redox-sensitive cell signaling. We propose that redox imbalances caused by VPA exposure result in improper cellular differentiation that may contribute to FVS. In undifferentiated P19 mouse embryonal carcinoma cells treated with VPA, glutathione disulfide (GSSG) concentrations were higher and the glutathione (GSH)/GSSG redox potential (Eh) was more oxidizing compared to vehicle-treated control cells, both of which are indications of potential intracellular oxidative stress. Interestingly, VPA had no effect on GSH or GSSG levels in differentiated P19 neurons. Undifferentiated cells pretreated with 3H-1,2-dithiole-3-thione (D3T), an inducer of the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant response that combats cellular redox disruption, were protected from VPA-induced alterations to the GSH/GSSG system. To assess differential periods of susceptibility, P19 cells were exposed to VPA at various time points during their neuronal differentiation. Cells exposed to VPA early in the differentiation process did not undergo normal neurogenesis as measured by POU domain, class 5, transcription factor 1 (OCT4) and tubulin beta-3 chain (βIII-tubulin), markers of cell stemness and neuronal differentiation, respectively. Neurogenesis was improved with D3T pretreatments prior to VPA exposure. Furthermore, differentiating P19 cells treated with VPA exhibited increased protein oxidation that was diminished with D3T pretreatment. These findings demonstrate that VPA inhibits neurogenesis and propose NRF2-mediated redox homeostasis as a means to promote normal neuronal differentiation, thereby potentially decreasing the prevalence of FVS outcomes.

A novel derivative of picolinic acid induces endoplasmic reticulum stress-mediated apoptosis in human non-small cell lung cancer cells: synthesis, docking study, and anticancer activity

Thirteen new derivatives of picolinic acid (4–7) were designed and synthesized from the starting parent molecule, picolinic acid. The new compounds were characterized by ATR-FTIR, 1HNMR, and CHNS analysis. A molecular docking study was performed to evaluate the binding affinity of the synthesized compounds toward EGFR kinase domain that indicated occupation of the critical site of EGFR kinase pocket and excellent positioning of the compounds in the pocket. The cytotoxic activity of the compounds against two human cancer cell lines (A549 and MCF-7), the non-tumorigenic MCF10A cell line, and white blood cells (WBC) was evaluated using the MTT assay. Compound 5 showed anticancer activity against A549 lung cancer cells (IC50 = 99.93 µM) but not against MCF-7 breast cancer cells or normal cells. Compound 5 mediated cytotoxicity in A549 lung cancer cells by inducing apoptotic cell death, as suggested by fragmented nuclei after DAPI staining, and agarose gel electrophoresis. Moreover, compound 5 triggered the activation of caspases 3, 4 and 9. However, compound 5 treatment did not affect the release of cytochrome c from the mitochondria to the cytosol, as compared to the vehicle-treated control cells. Nevertheless, compound 5-treated cells reported greater release of smac/DIABLO to the cytosol. In the same context, both compound 5 and thapsigargin (specific inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)) enhanced eIF2 phosphorylation, reflecting the activation of the atypical ER stress pathway and the potential applicability of compound 5 in lung cancer treatment.

Mechanistic Targets of Diallyl Trisulfide in Human Breast Cancer Cells Identified by RNA-seq Analysis.

Diallyl trisulfide (DATS), a metabolic by-product of processed garlic, is highly effective in inhibiting growth of human breast cancer cells in vitro and in vivo, but the underlying mechanisms are still not fully understood. In this study, we performed RNA-seq analyses using luminal-type (MCF-7) and basal-like (MDA-MB-231) human breast cancer cells to identify mechanistic targets of DATS. The Reactome Pathway Analysis revealed upregulation of genes associated with SLIT/ROBO tumor suppressor signaling following DATS treatment in both MCF-7 and MDA-MB-231 cells. However, the expression of SLIT2 and ROBO1 proteins or their downstream target C-X-C motif chemokine receptor 4 was not affected by DATS treatment in both cell lines. The Reactome as well as the Gene Ontology Pathways Analyses of the RNA-seq data from DATS-treated cells indicated downregulation of genes associated with G2/M phase cell cycle arrest in comparison with vehicle-treated control cells. Consistent with the RNA-seq data, DATS treatment caused a significant increase in the fraction of the G2/M population in both cell lines when compared to corresponding control cells. In addition, Ser10 phosphorylation of histone H3, a mitotic marker, was also increased significantly following DATS treatment in MCF-7 and MDA-MB-231 cells. These results indicate that while SLIT/ROBO signaling is not affected by DATS treatment, cell cycle arrest likely contributes to the antitumor effect of this phytochemical.

Tankyrase inhibitor XAV-939 enhances osteoblastogenesis and mineralization of human skeletal (mesenchymal) stem cells

Tankyrase is part of poly (ADP-ribose) polymerase superfamily required for numerous cellular and molecular processes. Tankyrase inhibition negatively regulates Wnt pathway. Thus, Tankyrase inhibitors have been extensively investigated for the treatment of clinical conditions associated with activated Wnt signaling such as cancer and fibrotic diseases. Moreover, Tankyrase inhibition has been recently reported to upregulate osteogenesis through the accumulation of SH3 domain-binding protein 2, an adaptor protein required for bone metabolism. In this study, we investigated the effect of Tankyrase inhibition in osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSCs). A Tankyrase inhibitor, XAV-939, identified during a functional library screening of small molecules. Alkaline phosphatase activity and Alizarin red staining were employed as markers for osteoblastic differentiation and in vitro mineralized matrix formation, respectively. Global gene expression profiling was performed using the Agilent microarray platform. XAV-939, a Tankyrase inhibitor, enhanced osteoblast differentiation of hBMSCs as evidenced by increased ALP activity, in vitro mineralized matrix formation, and upregulation of osteoblast-related gene expression. Global gene expression profiling of XAV-939-treated cells identified 847 upregulated and 614 downregulated mRNA transcripts, compared to vehicle-treated control cells. It also points towards possible changes in multiple signaling pathways, including TGFβ, insulin signaling, focal adhesion, estrogen metabolism, oxidative stress, RANK-RANKL (receptor activator of nuclear factor κB ligand) signaling, Vitamin D synthesis, IL6, and cytokines and inflammatory responses. Further bioinformatic analysis, employing Ingenuity Pathway Analysis identified significant enrichment in XAV-939-treated cells of functional categories and networks involved in TNF, NFκB, and STAT signaling. We identified a Tankyrase inhibitor (XAV-939) as a powerful enhancer of osteoblastic differentiation of hBMSC that may be useful as a therapeutic option for treating conditions associated with low bone formation.

SAT-311 Role of SREBP in Regulation of Corticotroph Tumor ACTH Secretion and Cell Proliferation

Cushing Disease (CD) is a life-threatening condition with suboptimal medical treatment. To identify drugs that not only inhibit ACTH secretion to attain eucortisolemia but also inhibit tumor growth, we conducted a high throughput screen employing a novel “gain of signal” ACTH AlphaLISA assay. From a kinase inhibitor library containing 430 compounds, we identified the dual PI3K/HDAC inhibitor, CUDC-907, as a potent inhibitor of both in vitro and in vivo corticotroph tumor ACTH secretion and growth. By stepwise comparison of CUDC-907 with mono-functional PI3K and HDAC inhibitors, we demonstrated that CUDC-907 exerts its inhibitory effect on ACTH secretion primarily through its inhibition of HDAC activity at the POMC transcriptional level; while PI3K-mediated inhibition of corticotroph cell viability further contributes to reduced ACTH secretion. We also used RNA-seq to characterize the global transcriptiome changes associated with CUDC-907 treatment. Hierarchical clustering showed that 1432 differentially expressed genes (DEGs, p≤0.05 and fold-change≥1.5) were altered by CUDC-907 treatment in comparison to the vehicle-treated control cells. Gene ontology (GO) analysis of 456 downregulated and 976 upregulated DEGs revealed that the most enriched biological processes were cholesterol biosynthesis (GO:0006695, p=1.977e-17) and the type I interferon signaling pathway (GO: 0060337, p=4.928e-7) respectively. Further analysis demonstrated downregulation of the membrane-bound transcription factor sterol regulatory element binding proteins (SREBPs). Downregulation of SREBP-2 by CUDC-907 as well as the several other target enzymes in the cholesterol biosynthesis and uptake pathway including IDI2, NSDHL, MVD, and HMGCR, was confirmed by real-time PCR. To further characterize a role for SREBP-2 in regulation of corticotroph tumor ACTH secretion and proliferation, we employed siRNA targeting endogenous SREBP-2 (SREBP-2 mRNA, Control vs. siRNA 1±0.03 vs. 0.6±0.08, p<0.05), and demonstrated that knockdown of SREBP-2 not only inhibited POMC mRNA expression (POMC mRNA, 1±0.03 vs. 0.7±0.01, p<0.01), and ACTH secretion (ACTH (ng/mL) 29±0.4 vs. 23±0.3, p<0.005), but also suppressed cell proliferation (Relative Proliferation Rate, 1±0.01 vs. 0.7±0.01, p<0.005). This was further confirmed by overexpression of cleaved mature SREBP-2, which led to increased POMC expression and cell proliferation. We demonstrate for the first time the role of the SREBP-mediated cholesterol biosynthesis pathway in regulation of corticotroph tumor POMC regulation and growth. Our studies identify SREBP and cholesterol biosynthesis as a therapeutic target in CD.

Iminosugars With Endoplasmic Reticulum α-Glucosidase Inhibitor Activity Inhibit ZIKV Replication and Reverse Cytopathogenicity in vitro.

Zika virus (ZIKV), a vector-borne virus of the family Flaviviridae, continues to spread and remains a significant global public health threat. Currently, there are no approved vaccines or antivirals against ZIKV. We investigated the anti-ZIKV ability of three iminosugars with endoplasmic reticulum α-glucosidase inhibitor (ER-AGI) activity, namely deoxynojirimycin (DNJ), castanospermine, and celgosivir. None of the three iminosugars showed any significant cytotoxicity in Vero or human microglia CHME3 cells when applied for 72 h at concentrations up to 100 μM. Iminosugar treatment of Vero or CHME3 cells prior to ZIKV infection resulted in significant inhibition of ZIKV replication over 48 h. Reduction in ZIKV replication in iminosugar-treated cells was not associated with any significant change in the expression levels of key antiviral genes. Following infection with three different strains of ZIKV, iminosugar-treated Vero or CHME3 cells showed no cell death, whereas vehicle-treated control cells exhibited 50–60% cell death at 72 h post-infection (hpi). While there was no significant difference in apoptosis between iminosugar-treated and control cells, iminosugar-treated cells exhibited a substantial reduction of necrosis at 72 hpi following ZIKV infection. In summary, iminosugars with ER-AGI activity inhibit ZIKV replication and significantly reduce necrosis without altering the antiviral gene expression and apoptosis of infected human cells. The results of this study strongly suggest that iminosugars are promising anti-ZIKV antiviral agents and such warrant further in vivo studies.

Hedgehog Signaling Inhibition by Smoothened Antagonist BMS-833923 Reduces Osteoblast Differentiation and Ectopic Bone Formation of Human Skeletal (Mesenchymal) Stem Cells.

Background Hedgehog (Hh) signaling is essential for osteoblast differentiation of mesenchymal progenitors during endochondral bone formation. However, the critical role of Hh signaling during adult bone remodeling remains to be elucidated. Methods A Smoothened (SMO) antagonist/Hedgehog inhibitor, BMS-833923, identified during a functional screening of a stem cell signaling small molecule library, was investigated for its effects on the osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSC). Alkaline phosphatase (ALP) activity and Alizarin red staining were employed as markers for osteoblast differentiation and in vitro mineralization capacity, respectively. Global gene expression profiling was performed using the Agilent® microarray platform. Effects on in vivo ectopic bone formation were assessed by implanting hMSC mixed with hydroxyapatite-tricalcium phosphate granules subcutaneously in 8-week-old female nude mice, and the amount of bone formed was assessed using quantitative histology. Results BMS-833923, a SMO antagonist/Hedgehog inhibitor, exhibited significant inhibitory effects on osteoblast differentiation of hMSCs reflected by decreased ALP activity, in vitro mineralization, and downregulation of osteoblast-related gene expression. Similarly, we observed decreased in vivo ectopic bone formation. Global gene expression profiling of BMS-833923-treated compared to vehicle-treated control cells, identified 348 upregulated and 540 downregulated genes with significant effects on multiple signaling pathways, including GPCR, endochondral ossification, RANK-RANKL, insulin, TNF alpha, IL6, and inflammatory response. Further bioinformatic analysis employing Ingenuity Pathway Analysis revealed significant enrichment in BMS-833923-treated cells for a number of functional categories and networks involved in connective and skeletal tissue development and disorders, e.g., NFκB and STAT signaling. Conclusions We identified SMO/Hedgehog antagonist (BMS-833923) as a powerful inhibitor of osteoblastic differentiation of hMSC that may be useful as a therapeutic option for treating conditions associated with high heterotopic bone formation and mineralization.

Abstract P2039: Nitric Oxide Inhibition Enhances the Protein Expression of TNF-α Receptor Type 1 in Cultured M-1 Cells With Characteristics of Renal Collecting Duct Epithelium

Oxidative stress and inflammation are key factors in the development of salt sensitive hypertension (SSH). Enhanced sodium reabsorption in the distal nephron, particularly the collecting duct (CD) plays a critical role in SSH development. Although the decrease in nitric oxide (NO) production (oxidative stress) and the increase in tumor necrosis factor-alpha (TNFα; pro-inflammatory cytokine) are common factors involved in SSH, the interactive role of these factors in modulating sodium reabsorption in CD cell is not yet clearly understood. Activation of TNF-α receptor type 1 (TNFR1) induces natriuresis by inhibiting ENaC, which is abundant in renal CD cells. The present study examined the effect of NO inhibition on TNFR1 protein expression in cultured M-1 cells (ATCC® CRL-2038™) that maintain the characteristics of renal cortical collecting duct cells. These cells were grown in DMEM-F12 growth medium containing 10% FBS and 1% Penstrep at 37 0 C, 5% CO2, 99% relative humidity up to 90% confluence. Then these cells were trypsinized and seeded into 100mm petri-dishes and treated with NO inhibitor, Nitro-L-arginine methyl ester (L-NAME; 0.5 mM) alone and in combination with a NO donor compound (DETA NONOate; 0.5 mM) along with a vehicle (PBS) treated group for 24 hours followed by harvesting and lysed with protease inhibitor-containing RIPA buffer. Extracted Protein concentration was determined by BCA assay kit, and a predetermined protein concentration was loaded in each well for SDS-PAGE. Immunoblots were detected for TNFR1 using primary antibodies for TNFR1 (19139; Abcam) and GAPDH. It was observed that TNFR1 expression significantly increases to 68±9.9 % (n=6) in L-NAME treated cells compared to vehicle treated control cells (n=4). This increase was prevented in cells co-treated with DETA NONOate (n=6). Immunofluorescence staining for TNFR1 protein in these treated cells also showed qualitatively similar findings as in western blot analysis. As TNFR1 mediates TNFα induced natriuretic response in the kidney, these data suggest that enhanced TNFR1 activity during NO inhibition in CD cells helps to minimize excessive sodium retention, and thus plays a counter-regulatory role in the development of SSH induced in oxidative stress conditions.

Stem cell library screen identified ruxolitinib as regulator of osteoblastic differentiation of human skeletal stem cells

BackgroundBetter understanding of the signaling pathways that regulate human bone marrow stromal stem cell (hBMSC) differentiation into bone-forming osteoblasts is crucial for their clinical use in regenerative medicine. Chemical biology approaches using small molecules targeting specific signaling pathways are increasingly employed to manipulate stem cell differentiation fate.MethodsWe employed alkaline phosphatase activity and staining assays to assess osteoblast differentiation and Alizarin R staining to assess mineralized matrix formation of cultured hBMSCs. Changes in gene expression were assessed using an Agilent microarray platform, and data normalization and bioinformatics were performed using GeneSpring software. For in vivo ectopic bone formation experiments, hMSCs were mixed with hydroxyapatite–tricalcium phosphate granules and implanted subcutaneously into the dorsal surface of 8-week-old female nude mice. Hematoxylin and eosin staining and Sirius Red staining were used to detect bone formation in vivo.ResultsWe identified several compounds which inhibited osteoblastic differentiation of hMSCs. In particular, we identified ruxolitinib (INCB018424) (3 μM), an inhibitor of JAK-STAT signaling that inhibited osteoblastic differentiation and matrix mineralization of hMSCs in vitro and reduced ectopic bone formation in vivo. Global gene expression profiling of ruxolitinib-treated cells identified 847 upregulated and 822 downregulated mRNA transcripts, compared to vehicle-treated control cells. Bioinformatic analysis revealed differential regulation of multiple genetic pathways, including TGFβ and insulin signaling, endochondral ossification, and focal adhesion.ConclusionsWe identified ruxolitinib as an important regulator of osteoblast differentiation of hMSCs. It is plausible that inhibition of osteoblast differentiation by ruxolitinib may represent a novel therapeutic strategy for the treatment of pathological conditions caused by accelerated osteoblast differentiation and mineralization.

Comparative Gene Expression Analysis in WM164 Melanoma Cells Revealed That β-β-Dimethylacrylshikonin Leads to ROS Generation, Loss of Mitochondrial Membrane Potential, and Autophagy Induction.

Skin cancer is currently diagnosed as one in every three cancers. Melanoma, the most aggressive form of skin cancer, is responsible for 79% of skin cancer deaths and the incidence is rising faster than in any other solid tumor type. Previously, we have demonstrated that dimethylacrylshikonin (DMAS), isolated from the roots of Onosma paniculata (Boraginaceae), exhibited the lowest IC50 values against different tumor types out of several isolated shikonin derivatives. DMAS was especially cytotoxic towards melanoma cells and led to apoptosis and cell cycle arrest. In this study, we performed a comprehensive gene expression study to investigate the mechanism of action in more detail. Gene expression signature was compared to vehicle-treated WM164 control cells after 24 h of DMAS treatment; where 1192 distinct mRNAs could be identified as expressed in all replicates and 89 were at least 2-fold differentially expressed. DMAS favored catabolic processes and led in particular to p62 increase which is involved in cell growth, survival, and autophagy. More in-depth experiments revealed that DMAS led to autophagy, ROS generation, and loss of mitochondrial membrane potential in different melanoma cells. It has been reported that the induction of an autophagic cell death represents a highly effective approach in melanoma therapy.

Effect of DA-9801 on the Expression of Drug-metabolizing Enzymes and Transporters in Human Hepatocytes

DA-9801, a mixed extract of Dioscoreae japonica and Dioscorea nipponica Makino, is awaiting phase 3 clinical study for the prevention or treatment of diabetic neuropathy in USA. To investigate whether DA-9801 acts as a perpetrator in drug interactions or not, we evaluated the mRNA induction of phase I and II drug metabolizing enzymes, including cyto-chrome P450 (CYP), UDP-glucuronosyltransferases (UGT) and sulfotransferases (SULT), and drug transporters. The mRNA levels of phase I and II enzymes and transporters were analyzed by real-time PCR using three different cryo-preserved human hepatocytes using specific primer and probe primer sets. We found that no significant alteration of the mRNA expression was observed in phase I enzymes including CYP1A2, CYP3A4, CYP2B6, CYP2C8, and CYP2C9, phase II enzymes such as UGT1A1, UGT1A4, UGT1A9, UGT2B7 and SULT2A1, and drug transporters including p-glycoprotein, MRP2, BCRP and OCT1 by DA-9801 treatment compared with vehicle treated control cells. Moreover, we found that CYP1A2-catalyzed phenacetin O-deethylase, CYP2B6-catalyzed bupropion hydroxylase, and CYP3A4-catalyzed midazolam 1’-hydroxylase activities were not interfered by DA-9801 in cryopreserved human hepatocytes. These results suggested that DA-9801 might not cause pharmacokinetic-based drug interactions with concurrent drugs which are the substrates of these drug metabolizing enzymes and transporters in humans.

Abstract 3269A: 2-Hydroxyflavanone: A novel estrogen receptor alpha downregulator with potent antitumor effect in breast cancer

Abstract Breast cancer (BC) remains one of the major causes of cancer deaths in women. 2Hydroxyflavanone (2HF), a constituent of citrus fruits, inhibits growth of human breast cancer cell lines in culture. The present study was undertaken to determine in vivo efficacy of 2HF against MDAMB231 human breast cancer xenografts. The 2HF administration retarded growth of MDAMB231 cells subcutaneously implanted in female nude mice without causing weight loss or any other side effects. The 2HF mediated suppression of MDAMB231 xenograft growth correlated with reduced cell proliferation as revealed by immunohistochemical analysis for Ki67 expression. Analysis of the vasculature in the tumors from 2HF treated mice indicated smaller vessel area compared with control tumors based on immunohistochemistry for angiogenesis marker CD31. The 2HF mediated inhibition of angiogenesis in vivo correlated with downregulation of vascular endothelial growth factor (VEGF) level in the tumor. Consistent with these results, 2HF treatment suppressed cell cycle regulators levels in cultured MDAMB231 cells. Moreover, the 2HF treated MDAMB231 cells exhibited reduced capacity for migration compared with vehicle-treated control cells. In addition, 2HF administration elicits apoptotic response as judged by annexin V and PI staining and TUNEL assay. 2HF treatment reduced the levels of ER alpha in ER positive MCF7 breast cancer cells. In conclusion, the present study demonstrates in vivo anticancer efficacy of 2HF against MDAMB231 xenografts in association with reduced cell proliferation and suppression of neovascularization. Our results indicate that 2HF is a promising anticancer agent for breast cancer due to its ability to collectively inhibit major regulators of breast cancer including RLIP76, ER alpha, and HER2. Interestingly, a spontaneously immortalized non-tumorigenic normal human mammary epithelial cell line (MCF10A) is significantly more resistant to 2HF mediated apoptosis compared with breast cancer cells, demonstrating selectivity of 2HF towards malignant breast cells. These preclinical observations merit clinical investigation to determine efficacy of 2HF against human breast cancers. (This work was supported in part by the Department of Defense grant W81XWH 16 1 0641. Funding from the Beckman Research Institute of City of Hope is also acknowledged). Citation Format: Sharad S. Singhal, Shireen Chikara, Lokesh Nagaprashantha, Jyotsana Singhal, David Horne, Sanjay Awasthi. 2-Hydroxyflavanone: A novel estrogen receptor alpha downregulator with potent antitumor effect in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3269A.

1,25-Dihydroxycholecalciferol (calcitriol) modifies uptake and release of 25-hydroxycholecalciferol in skeletal muscle cells in culture

The major circulating metabolite of vitamin D3, 25-hydroxycholecalciferol [25(OH)D], has a remarkably long half-life in blood for a (seco)steroid. Data from our studies and others are consistent with the hypothesis that there is a role for skeletal muscle in the maintenance of vitamin D status. Muscle cells internalise vitamin D-binding protein (DBP) from the circulation by means of a megalin/cubilin plasma membrane transport mechanism. The internalised DBP molecules then bind to actin and thus provide an intracellular array of high affinity binding sites for its specific ligand, 25(OH)D. There is evidence that the residence time for DBP in muscle cells is short and that it undergoes proteolytic degradation, releasing bound 25(OH)D. The processes of internalisation of DBP and its intracellular residence time, bound to actin, appear to be regulated. To explore whether 1,25-dihydroxycholecalciferol (calcitriol) has any effect on this process, cell cultures of myotubes and primary skeletal muscle fibers were incubated in a medium containing 10−10M calcitriol but with no added DBP. After 3h pre-incubation with calcitriol, the net uptake of 25(OH)D by these calcitriol-treated cells over a further 4h was significantly greater than that in vehicle-treated control cells. This was accompanied by a significant increase in intracellular DBP protein. However, after 16h of pre-incubation with calcitriol, the muscle cells showed a significantly depressed ability to accumulate 25(OH)D compared to control cells over a further 4 or 16hours. These effects of pre-incubation with calcitriol were abolished in fibers from VDR-knockout mice. The effect was also abolished by the addition of 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), which inhibits chloride channel opening. Incubation of C2 myotubes with calcitriol also significantly reduced retention of previously accumulated 25(OH)D after 4 or 8h. It is concluded from these in vitro studies that calcitriol can modify the DBP-dependent uptake and release of 25(OH)D by skeletal muscle cells in a manner that suggests some inducible change in the function of these cells.

Detection and Quantification of Nuclear Morphology Changes in Apoptotic Cells by Fluorescence Microscopy and Subsequent Analysis of Visualized Fluorescent Signals.

Apoptosis results in specific and stage-dependent morphological alterations of the cell nucleus, including pyknosis and cell shrinking. The experimental investigation of apoptotic processes is still challenging and routinely based on the assessment of molecular events like chromatin fragmentation and caspase enzyme activity. Alternatively, the establishment of a fluorescence microscopy nuclear morphology assay would provide a simple and robust low-cost method for detection and quantification of apoptotic cascades. Model cell lines LNCaP and MDA-MB-231 were incubated in the presence of the apoptosis-inducer cycloheximide (CHX). After evaluation of apoptotic cascades by terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) assay, stained cell nuclei were analyzed regarding area, perimeter, major and minor axis, as well as brightness of nuclear fluorescence signal. When compared to vehicle-treated control cells, administration of CHX led to significantly reduced cell growth and elevated rates of chromatin fragmentation of both cell lines as shown by cell counting and TUNEL assay, respectively. These apoptotic effects were accompanied by apoptosis-specific modulations of the nuclei demonstrated by diminished nuclear morphology parameters, such as area, perimeter, major and minor axis, as well as elevated levels of nuclear staining intensity. We present a computerized method for apoptosis detection and quantification using images of fluorescent dye-stained cell nuclei. The advantages of this nuclear morphology assay include the (i) ability to routinely assess apoptosis by a fast, highly reproducible low-cost technique, (ii) applicability of an experimental approach analyzing high numbers of single nuclei and (iii) detection of apoptosis in early, as well as late, stages of the apoptotic cascade.