IntroductionThe oxidative modification of LDL seems a key event in atherogenesis and may participate in inflammatory tissue injury. Our previous studies suggested that the process of LDL oxidation by activated human monocytes/macrophages required O-and activity of intracellular lipoxygenase. Herein, we studied the mechanisms involved in this oxidative modification of LDL. In this study, we used the human monocytoid cell line U937 to examine the role of Ca2+ in U937 cell-mediated lipid peroxidation of LDL. U937 cells were activated by opsonized zymosan.Removal of Ca2+ from cell culture medium by EGTA inhibited U937 cell-mediated peroxidation of LDL lipids. Therefore, Ca 2+ influx and mobilization were examined for their influence on U937 cell-mediated LDL lipid peroxidation. Ca2+ channel blockers nifedipine and verapamil blocked both Ca2+ influx and LDL lipid peroxidation by activated U937 cells. The inhibitory effects of nifedipine and verapamil were dose dependent. TMB-8 and ryanodine, agents known to prevent Ca2' release from intracellular stores, also caused a dose-dependent inhibition of LDL lipid peroxidation by activated U937 cells while exhibiting no effect on Ca2+ influx. Thus, both Ca2+ influx through functional calcium channels and Ca2+ mobilization from intracellular stores participate in the oxidative modification of LDL by activated U937 cells. 45Ca2+ uptake experiments revealed profound Ca2+ influx during the early stages of U937 cell activation, however, the Ca2+ ionophore 4-bromo A23187 was un- Oxidized LDL has been shown to be toxic to proliferating target cells in vitro, such as fibroblasts and endothelial cells (1,4,5). If activated monocytes/macrophages were able to oxidize LDL in vivo, the resultant toxic lipoproteins could mediate tissue injury in a variety of inflammatory responses. This may likely occur in the developing atherosclerotic lesion where lipid-laden foam cells of monocyte origin are surrounded by LDL accumulating in high concentration in the vascular interstitial space (5,6). Thus, it appears that phagocyte-derived free radicals, both superoxide anion and lipid peroxyl radicals, oxidize LDL and that the products oflipid peroxidation carried by oxidized LDL can effect more extensive or more distant tissue injury than that directly mediated by monocyte-derived free radicals.Although both the release ofreactive oxygen species by activated human monocytes/macrophages and the requirement for cellular lipoxygenases have been correlated with oxidation of LDL lipids, the precise mechanisms involved in the oxidation are not entirely understood. In particular, little is known regarding the intracellular signaling required for activated monocytes to mediate oxidation of LDL. Studies on the required intracellular signaling can help elucidate the specific mechanisms of activated monocyte-mediated oxidation of LDL and conversion of LDL to cytotoxin as well as suggest optimal means for intervening and preventing this process. tors, complement receptors, and as a stimulator of phagoc...
Objectives The goal of this study was to determine if a proprietary muscadine grape seed and skin extract (MGE) inhibits triple negative breast cancer (TNBC) metastasis and alters the gut microbiota. Methods 4T1 TNBC cells were injected into the mammary fat pad of 6-week-old female Balb/c mice. After 2 weeks, tumors were surgically removed and mice were placed into a control group (n = 8) or a treatment group that received 0.1 mg/mL total phenolics MGE (Piedmont R&D) in the drinking water (n = 8). Mice were sacrificed after 4 weeks; tissues and fecal samples were collected for analysis. Immunohistochemistry (Ki67, α-SMA) and hemotoxylin and eosin staining were used to quantify metastases using the inForm© 2.2 software. Gut microbial composition was determined by 16S rRNA sequencing and short chain fatty acids were detected by gas chromatography (Microbiome Insights). Data are expressed as means ± SEM using student's t-test. Results MGE reduced Ki67 cell positivity in the lungs and livers of mice, indicating reduced metastatic proliferation (9.3 ± 0.9% vs 6.2 ± 0.7% and 5.0 ± 1.5% vs 0.77 ± 0.2% cells, respectively; P < 0.01), and decreased cancer associated fibroblasts in the lungs (5.3 ± 1.0% vs 3.0 ± 0.5% cells; P < 0.05), which are associated with metastasis. MGE significantly reduced the number (4.7 ± 0.7 vs 2.2 ± 0.4 tumors/field; P < 0.01) and size (1358 ± 48 vs 1121 ± 47 pixels; P < 0.01) of liver metastases, resulting in decreased metastatic tumor burden (6656 ± 1220 vs 3096 ± 644 total area in pixels; P < 0.01). Attenuated TNBC metastasis correlated with MGE-induced changes in gut microbiota. Alpha diversity (4.15 ± 0.10 vs 4.51 ± 0.13 Shannon index; P < 0.05) and the Firmicutes to Bacteroidetes ratio (0.37 ± 0.07 vs 0.76 ± 0.12; P < 0.05) were significantly increased in MGE-treated mice, indicating enhanced microbial richness and increased energy harvest by the gut microbiome. Butyrate-producing bacteria, such as Ruminococcus, Butyricicoccus and Lachnospiraceae, were increased with MGE (P < 0.05) as well as the anti-inflammatory compound butyrate relative to other short-chain fatty acids (25.0 ± 2.7% vs 75.3 ± 15.5%; P < 0.01). Conclusions These data show that MGE attenuates TNBC metastasis in association with alterations in the gut microbiome, suggesting that MGE may be an effective treatment against TNBC metastatic progression. Funding Sources Chronic Disease Research Fund.
Tumor metastasis to the brain is a common complication of cancer, affecting 500,000 patients each year. Due to poor penetration of most chemotherapeutics into the brain, brain metastasis may occur even while systemic disease in under control. Despite aggressive treatments including radiation therapy, survival at 12 months following diagnosis is only 20%, underscoring the need for better means to prevent and/or treat brain metastases. Natural products have historically been a very successful source of new drugs. We are investigating the anti-cancer potential of a proprietary muscadine grape extract (MGE; from Piedmont Research & Development Corp.) on breast cancer cell lines that are metastatic to the brain. In a colony formation assay, MGE (10 μg/mL) decreased clonogenic survival of 4T1.luc2.Br5 cells by 50% (n=3, p<0.0001). The Eo771.luc.Br5 cells were more sensitive to MGE, showing 33% inhibition at 5 μg/mL (n=3, p<0.001) and 91% inhibition at 7.5 μg/mL (n=3, p<0.0001). We also assessed response to the combined treatment of MGE and ionizing radiation (IR). While MGE did not sensitize cells to IR, combined administration of MGE and IR resulted in decreased clonogenic survival compared to either modality alone for both cell lines (n=3-4 experiments, p<0.01), suggesting an additive effect. Mechanistically, acute exposure (24 h) to 10 μg/mL MGE induced apoptosis in 4T1.luc2.Br5 cells as evidenced by PARP cleavage and detection of activated cleaved caspase 3. Further, ERK1/2 activation was decreased 68% at this time point (n=3, p<0.05). In contrast, although Eo771.luc.Br5 cells are more sensitive to clonogenic inhibition, no PARP cleavage, caspase 3 cleavage, or ERK1/2 inhibition was observed. Analysis of cyclin D1, a major regulator of cell cycle progression, identified >80% decrease following MGE treatment (24 h, 10 μg/mL) in both cell lines. Together these data suggest that MGE inhibits breast cancer brain metastatic cell proliferation by multiple mechanisms, including inhibition of clonogenic growth through induction of apoptosis and/or decreased cell cycle progression, suggesting that MGE may have utility in the treatment of breast cancer brain metastasis. Citation Format: Wenhong Chen, Patricia Gallagher, Ann Tallant, Linda J. Metheny-Barlow. Muscadine grape extract inhibits breast cancer brain metastatic cells by multiple mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5125. doi:10.1158/1538-7445.AM2017-5125
Introduction and HypothesisEllagitannins are abundant in various foods including fruits, nuts and wines, as well as plant‐based supplements. Urolithin A is derived from ellagitannins metabolism by the microbiome, is absorbed by the gut epithelium and may significantly contribute to the biological actions of ellagic acid. Previous studies demonstrate renal protective effects of both ellagic acid and urolithin A; however, the cellular mechanisms that underlie the benefits of urolithin A are unclear. Activation of the renal TGFβ‐PAI‐1 pathway is a key cellular event that contributes to fibrosis, oxidative stress and progressive kidney injury. Therefore, the current study assessed the effects of urolithin A on TGFβ‐induced stimulation of PAI‐1 in renal proximal tubule (PT) cells. We hypothesized that urolithin A would present protective effects to attenuate PAI‐1 expression in PT cells and exhibit greater potency than the parent compound ellagic acid.ResultsWe initially determined an optimal dose of TGFβ (0.1 to 10 ng/ml) to stimulate PAI‐1 release in the NRK‐52e cell line. Cells were maintained in serum free conditions for 48 hours prior to TGFβ treatment. At a concentration of 5 ng/ml, TGFβ increased PAI‐1 approximately 5‐fold (480 ± 35%, n=4; p<0.01) above unstimulated or basal levels following a 24 hour exposure. As expected, pretreatment with the TGFβ receptor (TGFβR) kinase inhibitor SB431542 (1 uM) abolished the increase in PAI‐1. Moreover, the EGFR kinase inhibitor AG1478 (1 μM) completely abrogated the PAI‐1 release in response to TGFβ and confirms earlier reports that TGFβ stimulation of PAI‐1 reflects the complete dependence on activation of the EGFR. In contrast, the cSRC kinase inhibitor SU6056 failed to block PAI‐1 release suggesting that TGFβ stimulates the ligand‐dependent transactivation of EGFR rather than direct cSRC activation of the EGFR. Similar to TGR and EGFR inhibition, pretreatment of the cells with urolithin A (40 μM) abolished the increase in PAI‐1 to TGFβ (5 ± 4% of basal release, n=4). Dose response studies of urolithin A revealed an ED50 of 22 ± 3 μM (n=3) to inhibit PAI‐1. Ellagic acid also inhibited TGFβ stimulation of PAI‐1 release, but was less potent (ED50 >50 μM) than Urolithin A.ConclusionWe conclude that the protective effects of urolithin A may involve inhibition of the TGFβ‐EGFR‐PAI‐1 pathway in renal epithelial cells. Although the precise inhibitory mechanisms are presently unknown, urolithin A may potentially target either TGFβR‐ or EGFR‐dependent kinases to abrogate PAI‐1 expression.Support or Funding InformationAHA Transformative Grant 18TPA34170522; Anonymous Contribution 15‐01401This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Introduction: Ellagitanins are highly abundant in various foods including fruits, nuts and wines, and particularly high in commercial muscadine grape-derived supplements. The microbiome metabolizes ellagitanins to urolithins. Urolithin A (UA), the final product of the microbiome processing pathway may contribute to the cardioprotective effects of ellagitanins; however, the cellular signaling mechanisms of UA are unclear. Since the TGFβ-PAI-1 pathway contributes to fibrosis, cellular senescence and progressive kidney injury, we evaluated the effects of UA on TGFβ-induced stimulation of PAI-1 in rat renal proximal tubule cells (rPTCs - NRK52E). We hypothesize that UA may be an endogenous anti-fibrotic compound that attenuates the TGFβ-PAI-1 response. Results: TGFβ (5 ng/ml) treatment markedly increased PAI-1 levels 10-fold [43.9 ± 0.7 vs 3.5 ± 0.3 ng/ml; P<0.001, n=3] in the rPTCs at 24 hrs. The TGFβ receptor (TGFβR) kinase (ALK5) inhibitor SB525334 or UA abolished the increase in PAI-1 to TGFβ [SB: 1.3 ± 0.3; UA: 2.0 ± 0.4 ng/ml; P<0.001, n=3]; UA exhibits an IC50 of 3.1 μM to abrogate the TGFβ-PAI-1 response. Since the TGFβ pathway may transactivate the EGFR, treatment with the EGFR kinase inhibitors AG1478 (AG, 1 μM) and Lapatinib (LAP, 1 μM) also abrogated the PAI-1 response to TGFβ [AG: 3.3 ± 0.4; LAP: 6.2 ± 1.4 ng/ml; P<0.001, n=3]. Addition of EGF alone increased PAI-1 to a similar extent as TGFβ [46.6 ± 2.7 ng/ml; P<0.001, n=3], and treatment with UA or AG abolished the EGF-PAI-1 response [2.4 ± 0.1 and 1.3 ± 0.1 ng/ml, respectively; P<0.001, n=3]. Given the dependence on EGFR signaling for PAI-1, we determined whether UA directly attenuates EGFR stimulation. Indeed, UA treatment inhibited peak EGFR phosphorylation to EGF at 5 mins [10 μM: 67 ± 4%; 50 μM: 71 ± 5%; P<0.05, n=3], and reduced total EGFR expression at 24 hrs [10 μM: 27 ± 11%; 50 μM: 66 ± 7%; P<0.05, n=6]. UA was devoid of cytotoxic effects (LDH release). Conclusion: The present study is the first to establish that UA abrogates the TGFβ-EGFR-PAI-1 pathway via the inhibition of EGFR stimulation and expression. Moreover, the potency of UA to attenuate PAI-1 is comparable to its physiological levels. The microbiome product UA may convey anti-fibrotic actions within the kidney by targeting the EGFR system.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.