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.
Grignard reagents undergo conjugate addition to thiochromones catalyzed by copper salts to afford 2-substituted-thiochroman-4-ones, both 2-alkylthiochroman-4-ones and thioflavanones (2-arylthiochroman-4-ones), in good yields with trimethylsilyl chloride (TMSCl) as an additive. The best yields of 1,4-adducts can be attained with CuCN∙2LiCl as the copper source. Excellent yields of 2-alkyl-substituted thiochroman-4-ones and thioflavanones (2-aryl substituted) are attained with a broad range of Grignard reagents. This approach works well with both alkyl and aromatic Grignard reagents, thus providing a unified synthetic approach to privileged 2-substituted thiochroman-4-ones and a potential valuable precursor for further synthetic applications towards many pharmaceutically active molecules. The use of commercially available and/or readily prepared Grignard reagents will expedite the synthesis of a large library of both 2-alkyl substituted thiochroman-4-ones and thioflavanones for additional synthetic applications.
Background Triple negative breast cancer (TNBC) has a high propensity to metastasize and no treatments are available to slow or prevent metastatic progression. The goal of this study is to determine whether a proprietary high-polyphenol content muscadine grape extract (MGE) inhibits TNBC metastasis. 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 (n=8) or treatment group that received 0.1 mg/mL total phenolics MGE in the drinking water (n=8) for 4 weeks. Immunohistochemistry (Ki67, α-SMA) and hemotoxylin and eosin staining were used to quantify metastases. Gut microbial composition was determined by 16S rRNA sequencing and short chain fatty acids (SCFAs) were detected by gas chromatography. MDA-MB-231, BT-549 and 4T1 TNBC cell motility and cytoskeletal organization was assessed in vitr o by scratch wound migration and confocal microscopy, respectively. Data were evaluated by student’s t -test. Results MGE reduced metastatic proliferation in mouse lungs (33.3%) and livers (58.3%) and decreased the number (51.1%) and size (17.4%) of liver metastases, resulting in a 55.7% reduction in metastatic tumor burden ( P < 0.01). Serum IL-6 was reduced 99.6% in MGE-treated mice ( P = 0.06). MGE attenuated migration, altered cytoskeletal organization, and reduced RHAMM expression in TNBC cells ( P < 0.05). The gut microbiota, a mediator of polyphenolic bioactivities, was altered significantly in MGE-treated mice; MGE increased the alpha diversity (7.14%), Firmicutes/Bacteroidetes ratio (2-fold), relative abundance of butyrate-producing genera, and butyrate (3-fold) ( P < 0.05). Butyrate inhibited 4T1 cell proliferation and migration, suggesting butyrate contributes to MGE’s anti-metastatic activities ( P < 0.05). Conclusion Our results indicate that MGE may be an effective adjuvant therapy to reduce TNBC metastatic progression.
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