Cholesterol neutralized vemurafenib treatment by promoting melanoma stem-like cells via its metabolite 27-hydroxycholesterol.

Vemurafenib has been used as first-line therapy for unresectable or metastatic melanoma with BRAFV600E mutation. However, overall survival is still limited due to treatment resistance after about one year. Therefore, identifying new therapeutic targets for melanoma is crucial for improving clinical outcomes. In the present study, we found that lowering intracellular cholesterol by knocking down DHCR24, the limiting synthetase, impaired tumor cell proliferation and migration and abrogated the ability to xenotransplant tumors. More importantly, administration of DHCR24 or cholesterol mediated resistance to vemurafenib and promoted the growth of melanoma spheroids. Mechanistically, we identified that 27-hydroxycholesterol (27HC), a primary metabolite of cholesterol synthesized by the enzyme cytochrome P450 27A1 (CYP27A1), reproduces the phenotypes induced by DHCR24 or cholesterol administration and activates Rap1-PI3K/AKT signaling. Accordingly, CYP27A1 is highly expressed in melanoma patients and upregulated by DHCR24 induction. Dafadine-A, a CYP27A1 inhibitor, attenuates cholesterol-induced growth of melanoma spheroids and abrogates the resistance property of vemurafenib-resistant melanoma cells. Finally, we confirmed that the effects of cholesterol on melanoma resistance require its metabolite 27HC through CYP27A1 catalysis, and that 27HC further upregulates Rap1A/Rap1B expression and increases AKT phosphorylation. Thus, our results suggest that targeting 27HC may be a useful strategy to overcome treatment resistance in metastatic melanoma.

Postbiotic of Pediococcus acidilactici GQ01, a Novel Probiotic Strain Isolated from Natural Fermented Wolfberry, Attenuates Hyperuricaemia in Mice through Modulating Uric Acid Metabolism and Gut Microbiota.

Hyperuricaemia (HUA) is a disorder of purine metabolism, which manifests itself as an increase in uric acid production and a decrease in uric acid excretion, as well as a change in the structure of the intestinal microbiota. Most of the drugs currently used to treat HUA have significant side effects, and it is essential to find a treatment for HUA that is free of side effects. In this study, a novel strain, Pediococcus acidilactici GQ01, was screened from natural fermented wolfberry. The effects of both live bacteria GQ01 and its heat-killed G1PB postbiotic on HUA were investigated. The results showed that both probiotic GQ01 and G1PB postbiotics could effectively decrease blood uric acid, creatinine, and urea nitrogen levels in the HUA mice model. P. acidilactici GQ01 was more effective in inhibiting ADA activity, while G1PB postbiotics was more effective in inhibiting XOD activity. Meanwhile, GQ01 and G1PB were able to ameliorate liver and kidney tissue injury, upregulate the expression of ABCG2 in kidney and XOD gene in liver, downregulate the protein expression of URAT1 and GLUT9 in kidney, and therefore reduce the value of blood uric acid by decreasing the uric acid reabsorption and increasing the excretion of uric acid. Additionally, both probiotics and postbiotics could regulate the intestinal microbiota structure of HUA mice, so as to bring the dysfunctional intestinal composition back to normal. Furthermore, P. acidilactici GQ01 and G1PB postbiotics can increase the levels of acetic acid, propionic acid, and butyric acid in the intestinal tract, improve the intestinal function, and maintain the healthy homeostatic state of the intestinal tract. In summary, P. acidilactici GQ01 and its G1PB postbiotics may be developed as functional food or drug materials capable of treating HUA.

Combinatorial transcriptional regulation of HEB/ZEB1/ASCL1 and MYBL2 on Ras/ErbB signaling.

Gene expression is tightly regulated by transcription factors (TFs) which play an important role in development and tumorigenesis. Abnormal transcriptional regulation leads to oncogene activation or tumor suppressor inhibition, thus promoting the occurrence and progression of tumors. MYBL2 (alias B-Myb), a ubiquitously expressed transcription factor of the MYB family, is a nuclear protein involved in cell cycle progression and overexpressed and associated with poor patient outcomes in numerous cancer entities. However, the further effectors of the MYBL2 downstream transcriptional network mediating its cancer-promoting properties remain not well elaborated. Here, we systemic investigated the global MYBL2 targets base on ChIP-seq data from melanoma, breast cancer, lung carcinoma, and liver cancer. Functional enrichment and further validation of MYBL2 downstream binding targets on melanoma cells demonstrated that genes in the Ras and ErbB signaling pathways were regulated by MYBL2. Moreover, when integrating breast cancer, lung carcinoma and liver cancer data, we identified HEB, ZEB1 and ASCL1 colocalized on Ras/ErbB signaling gene locus with MYBL2, indicating the regulatory complex on activating oncogenic expression. Taken together, this study provides a reference for a better understanding of the MYBL2 regulatory mechanism in tumorigenesis.

Downstream Regulatory Network of MYBL2 Mediating Its Oncogenic Role in Melanoma.

The transcription factor MYBL2 is widely expressed in proliferating cells. Aberrant expression of MYBL2 contributes to tumor malignancy and is associated with poor patient prognosis. However, the downstream transcriptional network that mediates its oncogenic properties remains elusive. In the present study, we observed that MYBL2 was overexpressed in malignant and metastatic melanoma patient samples and that the high expression level of MYBL2 was significantly associated with poor prognosis. A loss-of-function study demonstrated that MYBL2 depletion significantly decreased cell proliferation and migration and prevented cell cycle progression. We also determined that MYBL2 promoted the formation of melanoma stem-like cell populations, indicating its potential as a therapeutic target for treating resistant melanoma. Mechanistically, we constructed an MYBL2 regulatory network in melanoma by integrating RNA-seq and ChIP-seq data. EPPK1, PDE3A, and FCGR2A were identified as three core target genes of MYBL2. Importantly, multivariate Cox regression and survival curve analysis revealed that PDE3A and EPPK1 were negatively correlated with melanoma patient survival; however, FCGR2A was positively correlated with patient survival. Overall, our findings elucidate an MYBL2 regulatory network related to cell proliferation and cancer development in melanoma, suggesting that MYBL2 may be potentially targeted for melanoma diagnosis and treatment.

7-dehydrocholesterol suppresses melanoma cell proliferation and invasion via Akt1/NF-κB signaling.

Melanoma is the most lethal cutaneous cancer with a high metastatic rate worldwide, causing ~55,500 deaths annually. Although the selective B-Raf oncogene serine/threonine-kinase (BRAF) inhibitors, dabrafenib and vemurafenib, have been approved for the treatment of BRAF-mutant metastatic melanoma, the 5-year survival rate remains unfavorable due to acquired therapy resistance. Therefore, it is of great importance to develop alternative therapeutic drugs and uncover their mechanisms for the treatment of melanoma. 7-dehydrocholesterol (7-DHC) has been demonstrated to inhibit melanoma, but the mechanism is unclear. Therefore, the present study aimed to elucidate the mechanisms of the inhibitory effect of 7-DHC in melanoma cells via analyzing the proliferation, migration, apoptosis, cell cycle and transcriptional sequencing of melanoma cells treated with 7-DHC, as well as constructing a gene signature according to public data of patients with melanoma. In the present study, 7-DHC, the precursor of vitamin D3, was able to induce apoptosis and inhibit cell proliferation and invasion of melanoma cells in a dose-dependent manner. RNA sequencing of melanoma cells treated with different concentrations of 7-DHC revealed that, compared with untreated melanoma cells, 65 genes were downregulated, and genes involved in the regulation of NF-ĸB import into the nucleus and NF-ĸB signaling were significantly repressed. Consistently, the Akt kinase family was one of most common somatic mutation hotspots in patients with melanoma according to The Cancer Genome Atlas enrichment analysis. Furthermore, 7-DHC decreased the phosphorylation of Akt1-Ser473 rather than that of MEK1, and the decreased phosphorylation of Akt1 subsequently inhibited the translocation of free RELA proto-oncogene NF-κB subunit to the nucleus. Finally, by intersecting downregulated genes by 7-DHC treatment and upregulated genes in patients with melanoma, a 7-DHC gene signature was identified, which was negatively associated with the prognosis. Overall, the present results demonstrated that 7-DHC suppressed melanoma cell proliferation and invasion via the Akt1/NF-ĸB signaling pathway, and 7-DHC key target genes were negatively associated with the prognosis. These findings highlight the potential application of 7-DHC for the treatment of melanoma in the future.

Myristoleic acid produced by enterococci reduces obesity through brown adipose tissue activation.

OBJECTIVE: Dietary fibre has beneficial effects on energy metabolism, and the majority of studies have focused on short-chain fatty acids produced by gut microbiota. Ginseng has been reported to aid in body weight management, however, its mechanism of action is not yet clear. In this study, we focused on the potential modulating effect of ginseng on gut microbiota, aiming to identify specific strains and their metabolites, especially long-chain fatty acids (LCFA), which mediate the anti-obesity effects of ginseng. DESIGN: Db/db mice were gavaged with ginseng extract (GE) and the effects of GE on gut microbiota were evaluated using 16S rDNA-based high throughput sequencing. To confirm the candidate fatty acids, untargeted metabolomics analyses of the serum and medium samples were performed. RESULTS: We demonstrated that GE can induce Enterococcus faecalis, which can produce an unsaturated LCFA, myristoleic acid (MA). Our results indicate that E. faecalis and its metabolite MA can reduce adiposity by brown adipose tissue (BAT) activation and beige fat formation. In addition, the gene of E. faecalis encoding Acyl-CoA thioesterases (ACOTs) exhibited the biosynthetic potential to synthesise MA, as knockdown (KD) of the ACOT gene by CRISPR-dCas9 significantly reduced MA production. Furthermore, exogenous treatment with KD E. faecalis could not reproduce the beneficial effects of wild type E. faecalis, which work by augmenting the circulating MA levels. CONCLUSIONS: Our results demonstrated that the gut microbiota-LCFA-BAT axis plays an important role in host metabolism, which may provide a strategic advantage for the next generation of anti-obesity drug development.

Metabolite profiling of ginsenosides in rat plasma, urine and feces by LC-MS/MS and its application to a pharmacokinetic study after oral administration of Panax ginseng extract.

Panax ginseng is widely consumed as a functional food in the form of tea, powder, capsules, among others, and possesses a range of pharmacological activities including adaptogenic, immune-modulatory, anti-tumor, anti-aging and anti-inflammatory effects. The aim of this study was to identify and quantify the major ginsenosides and their metabolites in rat plasma, urine and feces after administration of P. ginseng extract using LC-MS/MS. We collected rat plasma samples at 0.5, 1, 2, 4, 8, 12, 24 and 48 h, and the amounts of urine and fecal samples accumulated in 24 h. Fourteen major ginsenosides and their metabolites were observed in fecal samples at high levels; however, low levels of 11 ginsenosides were detected in urine samples. The pharmacokinetics of the major ginsenosides and their metabolites was investigated in plasma. The results indicated that the maximum plasma concentration, time to maximum concentration and area under the curve of compound K were significantly greater than those of other ginsenosides. This study thus provides valuable information for drug development and clinical application of P. ginseng.

Biotransformation of Panax ginseng extract by rat intestinal microflora: identification and quantification of metabolites using liquid chromatography-tandem mass spectrometry.

BACKGROUND: In general, after Panax ginseng is administered orally, intestinal microbes play a crucial role in its degradation and metabolization process. Studies on the metabolism of P. ginseng by microflora are important for obtaining a better understanding of their biological effects. METHODS: In vitro biotransformation of P. ginseng extract by rat intestinal microflora was investigated at 37°C for 24 h, and the simultaneous determination of the metabolites and metabolic profile of P. ginseng saponins by rat intestinal microflora was achieved using LC-MS/MS. RESULTS: A total of seven ginsenosides were detected in the P. ginseng extract, including ginsenosides Rg1, Re, Rf, Rb1, Rc, Rb2, and Rd. In the transformed P. ginseng samples, considerable amounts of deglycosylated metabolite compound K and Rh1 were detected. In addition, minimal amounts of deglycosylated metabolites (ginsenosides Rg2, F1, F2, Rg3, and protopanaxatriol-type ginsenosides) and untransformed ginsenosides Re, Rg1, and Rd were detected at 24 h. The results indicated that the primary metabolites are compound K and Rh1, and the protopanaxadiol-type ginsenosides were more easily metabolized than protopanaxatriol-type ginsenosides. CONCLUSION: This is the first report of the identification and quantification of the metabolism and metabolic profile of P. ginseng extract in rat intestinal microflora using LC-MS/MS. The current study provided new insights for studying the metabolism and active metabolites of P. ginseng.

Comparative Analysis of the Rats' Gut Microbiota Composition in Animals with Different Ginsenosides Metabolizing Activity.

Following oral intake of Panax ginseng, major ginsenosides are metabolized to deglycosylated ginsenosides by gut microbiota before absorption into the blood. As the composition of gut microbiota varies between individuals, metabolic activities are significantly different. We selected 6 rats with low efficiency metabolism (LEM) and 6 rats with high efficiency metabolism (HEM) from 60 rats following oral administration of Panax ginseng extract, and analyzed their gut microbiota composition using Illumina HiSeq sequencing of the 16S rRNA gene. The components of gut microbiota between the LEM and HEM groups were significantly different. Between the 2 groups, S24-7, Alcaligenaceae, and Erysipelotrichaceae occupied most OTUs of the HEM group, which was notably higher than the LEM group. Furthermore, we isolated Bifidobacterium animalis GM1 that could convert the ginsenoside Rb1 to Rd. The result implies that these specific intestinal bacteria may dominate the metabolism of Panax ginseng.

Cloning and Characterization of Ginsenoside-Hydrolyzing ß-Glucosidase from Lactobacillus brevis That Transforms Ginsenosides Rb1 and F2 into Ginsenoside Rd and Compound K.

The ginsenoside-hydrolyzing ß-glucosidase gene (bgy2) was cloned from Lactobacillus brevis. We expressed this gene in Escherichia coli BL21(DE3), isolated the resulting protein, and then utilized the enzyme for the biotransformation of ginsenosides. The bgy2 gene contains 2,223 bp, and encodes a protein of 741 amino acids that is a member of glycosyl hydrolase family 3. ß-Glucosidase (Bgy2) cleaved the outer glucose moieties of ginsenosides at the C-20 position, and the inner glucose at the C-3 position. Under optimal conditions (pH 7.0, 30°C), we used 0.1 mg/ml Bgy2 in 20 mM sodium phosphate buffer (PBS) for enzymatic studies. In these conditions, 1.0 mg/ml ginsenoside Rb1 and ginsenoside F2 were converted into 0.59 mg/ml ginsenoside Rd and 0.72mg/ml compound K, with molar conversion productivities of 69% and 91%, respectively. In pharmaceutical and commercial industries, this recombinant Bgy2 would be suitable for producting ginsenoside Rd and compound K.

Biotransformation of gypenoside XVII to compound K by a recombinant ß-glucosidase.

OBJECTIVE: To study the ß-glucosidase gene (bgy1) from Lactobacillus brevis that was cloned and expressed in Escherichia coli BL21 (DE3) and then using it for the biotransformation of gypenoside XVII. RESULTS: The bgy1 gene consists of 2283 bp encoding 761 amino acids, with homology to the glycosyl hydrolase family-3 protein domain. The enzyme (Bgy1) hydrolyzed the glucose moieties at the C-3 position and the outer glucose moieties at the C-20 position of gypenoside XVII. Using 0.1 mg enzyme ml(-1) in 20 mM sodium phosphate buffer at 30 °C and pH 6.0, 1 mg gypenoside XVII ml(-1) was transformed into 0.58 mg compound K ml(-1) within 6 h, with a corresponding molar conversion yield of 89 %. CONCLUSION: The recombinant Bgy1 is considered potentially useful for the practical preparation of compound K.