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In this study, novel ergosterol peroxide (EP) derivatives were synthesized and evaluated to assess their antiproliferative activity against four human cancer cell lines (A549, HepG2, MCF-7, and MDA-MB-231). Compound 3g exhibited the most potent antiproliferative activity, with an IC50 value of 3.20 µM against MDA-MB-231. This value was 5.4-fold higher than that of the parental EP. Bioassay optimization further identified 3g as a novel glutaminase 1 (GLS1) inhibitor (IC50 = 3.77 µM). In MDA-MB-231 cells, 3g reduced the cellular glutamate levels by blocking the glutamine hydrolysis pathway, which triggered reactive oxygen species production and induced caspase-dependent apoptosis. Molecular docking indicated that 3g interacts with the reaction site of the variable binding pocket by forming multiple interactions with GLS1. In a mouse model of breast cancer, 3g showed remarkable therapeutic effects at a dose of 50 mg/kg, with no apparent toxicity. Based on these results, 3g could be further evaluated as a novel GLS1 inhibitor for triple-negative breast cancer (TNBC) therapy. [ABSTRACT FROM AUTHOR]

Summary: Salvianolic acids (SA), such as rosmarinic acid (RA), danshensu (DSS), and their derivative salvianolic acid B (SAB), etc. widely existed in Lamiaceae and Boraginaceae families, are of interest due to medicinal properties in the pharmaceutical industries. Hundreds of studies in past decades described that 4‐coumaroyl‐CoA and 4‐hydroxyphenyllactic acid (4‐HPL) are common substrates to biosynthesize SA with participation of rosmarinic acid synthase (RAS) and cytochrome P450 98A (CYP98A) subfamily enzymes in different plants. However, in our recent study, several acyl donors and acceptors included DSS as well as their ester‐forming products all were determined in SA‐rich plants, which indicated that previous recognition to SA biosynthesis is insufficient. Here, we used Salvia miltiorrhiza, a representative important medicinal plant rich in SA, to elucidate the diversity of SA biosynthesis. Various acyl donors as well as acceptors are catalysed by SmRAS to form precursors of RA and two SmCYP98A family members, SmCYP98A14 and SmCYP98A75, are responsible for different positions' meta‐hydroxylation of these precursors. SmCYP98A75 preferentially catalyses C‐3′ hydroxylation, and SmCYP98A14 preferentially catalyses C‐3 hydroxylation in RA generation. In addition, relative to C‐3′ hydroxylation of the acyl acceptor moiety in RA biosynthesis, SmCYP98A75 has been verified as the first enzyme that participates in DSS formation. Furthermore, SmCYP98A enzymes knockout resulted in the decrease and overexpression leaded to dramatic increase of SA accumlation. Our study provides new insights into SA biosynthesis diversity in SA‐abundant species and versatility of CYP98A enzymes catalytic preference in meta‐hydroxylation reactions. Moreover, CYP98A enzymes are ideal metabolic engineering targets to elevate SA content. [ABSTRACT FROM AUTHOR]

The aim of this study was to compare the expression of fucosyltransferase 8 (FUT8) in breast cancer tissue and to investigate the relationship between this marker with tumor progression and its applicability to differential diagnosis. An immunohistochemical study was performed for FUT8 using the tissue microarray technique. In addition, the mRNA and protein levels of FUT8 in the tissue were also tested by real-time PCR and Western blot. There was a significant difference in cytoplasmic expression of FUT8 between breast cancer tissue and matched normal tissue (p<0.001). The percent of FUT8 staining in breast cancer tissues ranging from negative, weak positive, positive and strong positive were 2.7%, 40.2%, 54% and 3.2%, respectively. High FUT8 protein expression correlated with lymphatic metastasis (p=0.008) and with stage status (p=0.039). We detected that reduced FUT8 expression correlated with disease-free survival (p=0.02) and overall survival (p=0.04) of breast cancer patients. Expression of FUT8 can stratify breast cancer tissue and may be considered a prognostic marker for breast cancer patients.

Objective: To construct the lentiviral expression vector of human TOX high mobility group box family member 3 (TOX3) gene and the MDA-MB-231 cell line which stably over-expresses TOX3 gene., Methods: TOX3 gene was synthesized by the gene synthesis method and amplified by PCR, and then cloned into pLVEF-1a/GFP-Puro vector to construct pLVEF-1a/GFP-Puro-TOX3 lentiviral vector. After restriction enzyme analysis and sequence identification, the lentiviral vector was packaged and the titer was detected. The human breast cancer MDA-MB-231 cells were transfected with the recombinant lentiviral vector and cultured selectively by puromycin to acquire stably transfected cells. MDA-MB-231 cells which expressed GFP were observed by fluorescence microcopy. And the expression levels of TOX3 mRNA and protein in transfected MDA-MB-231 cells were detected by real-time quantitative PCR(qRT-PCR) and Western blotting, respectively., Results: Restriction enzyme digestion and sequence analysis demonstrated that the lentiviral expression vectors of pLVEF-1a/GFP-Puro and pLVEF-1a/GFP-Puro-TOX3 were successfully constructed, and the viral titers were respectively 2×10(8) TU/mL and 1×10(8) TU/mL after lentiviral packaging. And after being transfected, more than 95% cells expressed GFP under a fluorescence microscope. The results of qRT-PCR and Western blotting showed that, when compared with the MDA-MB-231-NC negative control group, the expression of TOX3 mRNA and protein significantly increased in the MDA-MB-231-TOX3 group., Conclusion: The study successfully constructed lentiviral expression vector of TOX3 gene and obtained MDA-MB-231 cell line stably over-expressing TOX3 gene by transfection with the recombinant vector.