Fingolimod exerts in vitro anticancer activity against hepatocellular carcinoma cell lines via YAP/TAZ suppression

Hepatocellular carcinoma (HCC) remains a notably global health challenge with high mortality rates and poor prognosis. The deregulation of the Hippo signalling pathway, especially the overexpression and activation of downstream effector Yes-associated protein (YAP), has been demonstrated to result in the rapid malignant evolution of HCC. In this context, multiple efforts have been dedicated to targeting YAP for HCC therapy, but effective YAP inhibitors are still lacking. In this study, through a YAP-TEAD (8×GTIIC) luciferase reporter assay, we identified fingolimod, an immunomodulatory drug approved for the treatment of multiple sclerosis, as a novel YAP inhibitor. Fingolimod suppressed the proliferation of HCC cell lines by downregulating the protein levels as well as the transactivating function of YAP. Overall, our current study not only identifies fingolimod as a novel YAP-targeting inhibitor, but also indicates that this clinically-approved drug could be utilized as a potential and feasible therapeutic drug for HCC

Specific protein kinase C isoforms α and βI are involved in follicle-stimulating hormone-induced mouse follicle-enclosed oocytes meiotic resumption.

Protein kinase C (PKC) is involved in gonadotrophin-induced oocyte maturation. In the present study, we investigated the role of specific PKC isoforms in the process of follicle-stimulating hormone (FSH)-induced oocyte meiotic resumption. Small antral follicles (200-300 µm in diameter) were isolated from immature mice and cultured in vitro. FSH significantly induced follicle-enclosed oocytes (FEOs) meiotic resumption after 8 hr culture. However, the induced effect of FSH was dose-dependently inhibited by the specific PKC α and βI inhibitor Gö6976, and 100 nM Gö6976 completely blocked FSH function in oocyte meiotic resumption. Furthermore, FSH dramatically induced the expression of transcripts encoding epidermal growth factor (EGF)-like growth factors Areg, Btc, and Ereg mRNA levels, and up-regulated tyrosine phosphorylation level of EGF receptor (EGFR) in granulosa cells. Blocking the function of EGFR by AG1478 eliminated the effect of FSH-induced FEOs meiotic resumption, suggesting that FSH induced oocyte maturation through the activation of EGFR. FSH-induced phosphorylation of EGFR could also be inhibited by Gö6976. Next, we examined the effect of FSH on the expression and phosphorylation PKC α and βI. FSH induced the expression of PKC α at mRNA and protein level, and also up-regulated its phosphorylation level in granulosa cells after 8 hr culture. However, FSH had no effect on the expression of PKC βI but down-regulated its phosphorylation level. In conclusion, FSH-induced activation of PKC α alone, or together with the inactivation of PKC βI in granulosa cells, participates in mouse oocyte meiotic resumption, possibly by the activation of EGFR signaling pathway

Caloric restriction remodels the hepatic chromatin landscape and bile acid metabolism by modulating the gut microbiota

Abstract Background Caloric restriction (CR) has been known to promote health by reprogramming metabolism, yet little is known about how the epigenome and microbiome respond during metabolic adaptation to CR. Results We investigate chromatin modifications, gene expression, as well as alterations in microbiota in a CR mouse model. Collectively, short-term CR leads to altered gut microbial diversity and bile acid metabolism, improving energy expenditure. CR remodels the hepatic enhancer landscape at genomic loci that are enriched for binding sites for signal-responsive transcription factors, including HNF4α. These alterations reflect a dramatic reprogramming of the liver transcriptional network, including genes involved in bile acid metabolism. Transferring CR gut microbiota into mice fed with an obesogenic diet recapitulates the features of CR-related bile acid metabolism along with attenuated fatty liver. Conclusions These findings suggest that CR-induced microbiota shapes the hepatic epigenome followed by altered expression of genes responsible for bile acid metabolism

The effect of PKC α and βI inhibitor Gö6976 on FSH-induced mouse FEOs meiotic resumption.

<p>FEOs were cultured in MEM medium only, supplemented with FSH (0.05 IU/mL) and/or PKC α and βI inhibitor Gö6976 (1–1000 nM) for the indicated period. The percent of GVBD (including polar body 1) per total number of oocytes (% GVBD) were calculated. Oocytes that had degenerated were excluded. The similar demonstrations of statistical analysis were also used in the following Figures. Data were represented as mean percentage of GVBD ± SEM of at least three independent experiments. The median number of FEOs in each group was 150. (<b>A</b>) The kinetics of FSH-induced FEOs meiotic resumption. **<i>P</i><0.01 and ***<i>P</i><0.001 compared with each corresponding time point in the control groups. (<b>B</b>) Gö6976 dose-dependently inhibited the function of FSH. Groups with a different letter are significantly different (<i>P</i><0.05). G, Gö6976; F, FSH.</p

The effect of Gö6976 on the phosphorylation of EGFR during FSH-induced FEOs meiotic resumption.

<p>(<b>A</b>) Granulosa cells were collected at the indicated culture time, and analyzed by western blotting. Immunoblots were performed by phospho-EGF receptor antibody, and GADPH was served as an internal standard. Relative intensities were quantified using Gel-pro analyzer 4.0 software. Bars show the mean ± SEM of three independent experiments. Relative levels with a different letter are significantly different (<i>P</i><0.05). (<b>B</b>) FEOs were cultured in MEM medium supplemented with 0.05 IU/mL FSH, 0.1 µM Gö6976, 10 ng/mL EGF and or 100 ng/mL AR for 16 h, and the rate of GVBD was scored at the end of culture. Data were represented as mean percentage of GVBD ± SEM of three independent experiments. The median number of FEOs in each group was 120. Groups with a different letter are significantly different (<i>P</i><0.05). C, control; F, FSH; G, Gö6976.</p

The effect of FSH on the expression and phosphorylation of PKC βI.

<p>Follicles were cultured in MEM medium with or without FSH, and granulosa cells were collected at the indicated culture time. Immunoblots were performed by PKC βI (<b>A</b>) and PKCβI-phospho-specific Thr 642 (<b>B</b>) antibodies, respectively. GADPH was served as an internal standard. Relative intensities were quantified using Gel-pro analyzer 4.0 software. Bars show the mean ± SEM of three independent experiments. Relative levels with a different letter are significantly different (<i>P</i><0.05).</p

The effect of FSH on the expression and phosphorylation of PKCα.

<p>Follicles were cultured in MEM medium with or without FSH, and granulosa cells were collected at the indicated culture time. (<b>A</b>) FSH promoted the expression of <i>Prckα</i> mRNA. The mRNA level of <i>Prckα</i> was normalized to the level of <i>Rpl19</i>. Mean value of granulosa cells at 0 h was normalized to 1. The values indicated by different letters are significantly different (<i>P</i><0.05). (<b>B</b>) and (<b>C</b>) FSH promoted the expression and phosphorylation of PKCα. In each treatment, granulosa cells from 50 follicles cultured were lysated, separated by 15% SDS-PAGE, and analyzed by western blotting. Immunoblots were performed by PKCα antibody and PKCα-phospho-specific Tyr497 antibody, respectively. GADPH was served as an internal standard. Relative intensities were quantified using Gel-pro analyzer 4.0 software. Bars show the mean ± SEM of three independent experiments. Relative levels with a different letter are significantly different (<i>P</i><0.05).</p