Phenotypic and metabolomic characteristics of mouse models of metabolic associated steatohepatitis.

BACKGROUND: Metabolic associated steatohepatitis (MASH) is metabolic disease that may progress to cirrhosis and hepatocellular carcinoma. Mouse models of diet-induced MASH, which is characterized by the high levels of fats, sugars, and cholesterol in diets, are commonly used in research. However, mouse models accurately reflecting the progression of MASH in humans remain to be established. Studies have explored the potential use of serological metabolites as biomarkers of MASH severity in relation to human MASH. METHODS: We performed a comparative analysis of three mouse models of diet-induced MASH in terms of phenotypic and metabolomic characteristics; MASH was induced using different diets: a high-fat diet; a Western diet; and a high-fat, high-cholesterol diet. Liver cirrhosis was diagnosed using standard clinical approaches (e.g., METAVIR score, hyaluronan level, and collagen deposition level). Mouse serum samples were subjected to nuclear magnetic resonance spectroscopy-based metabolomic profiling followed by bioinformatic analyses. Metabolomic analysis of a retrospective cohort of patients with hepatocellular carcinoma was performed; the corresponding cirrhosis scores were also evaluated. RESULTS: Using clinically relevant quantitative diagnostic methods, the severity of MASH was evaluated. Regarding metabolomics, the number of lipoprotein metabolites increased with both diet and MASH progression. Notably, the levels of very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) significantly increased with fibrosis progression. During the development of diet-induced MASH in mice, the strongest upregulation of expression was noted for VLDL receptor. Metabolomic analysis of a retrospective cohort of patients with cirrhosis indicated lipoproteins (e.g., VLDL and LDL) as predominant biomarkers of cirrhosis. CONCLUSIONS: Our findings provide insight into the pathophysiology and metabolomics of experimental MASH and its relevance to human MASH. The observed upregulation of lipoprotein expression reveals a feedforward mechanism for MASH development that may be targeted for the development of noninvasive diagnosis.

Linoleate-pazopanib conjugation as active pharmacological ingredient to abolish hepatocellular carcinoma growth.

Small molecule compounds targeting multiple kinases involved in neoangiogenesis have shown survival benefits in patients with unresectable hepatocellular carcinoma (HCC). Nonetheless, despite the beneficial effects of multikinase inhibitors (MKIs), a lack of boosting adjuvant limits their objective response rate. Lipid conjugates have been used to improve delivery efficacy or pharmaceutical benefits for decades. However, the feasibility of utilizing lipid-drug conjugates (LDCs) in HCC regimens remains untested. In this study, oral feeding of linoleate-fluorescein isothiocyanate conjugates showed that the compound was well distributed in a spontaneous HCC mouse model. Therefore, a rationale design was developed for chemically synthesizing a linoleate-pazopanib conjugate (LAPC). The LAPC showed a significantly improved cytotoxicity compared to the parental drug pazopanib. Pazopanib's angiogenic suppressing signals were not observed in LAPC-treated HCC cells, potentially suggesting an altered mechanism of action (MOA). In an efficacy trial comparing placebo, oral pazopanib, and LAPC treatments in the hepatitis B virus transgene-related spontaneous HCC mouse model (HBVtg-HCC), the LAPC treatment demonstrated superior tumor ablating capacity in comparison to both placebo and pazopanib treatments, without any discernible systemic toxicity. The LAPC exposure is associated with an apoptosis marker (Terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) and an enhanced ferroptosis (glutathione peroxidase 4 [GPX4]) potential in HBVtg-HCC tumors. Therefore, the LAPC showed excellent HCC ablative efficacy with altered MOA. The molecular mechanisms of the LAPC and LDCs for HCC therapeutics are of great academic interest. Further comprehensive preclinical trials (e.g., chemical-manufacture-control, toxicity, distribution, and pharmacokinetics/pharmacodynamics) are expected.

Role of Ketogenic Diets in Multiple Sclerosis and Related Animal Models: An Updated Review.

Prescribing a ketogenic diet (KD) is a century-old dietary intervention mainly used in the context of intractable epilepsy. The classic KD and its variants regained popularity in recent decades, and they are considered potentially beneficial in a variety of neurological conditions other than epilepsy. Many patients with multiple sclerosis (MS) have attempted diet modification for better control of their disease, although evidence thus far remains insufficient to recommend a specific diet for these patients. The results of 3 pilot clinical trials of KD therapy for MS, as well as several related studies, have been reported in recent years. The preliminary findings suggest that KD is safe, feasible, and potentially neuroprotective and disease-modifying for patients with MS. Research on corresponding rodent models has also lent support to the efficacy of KD in the prevention and treatment of experimental autoimmune encephalomyelitis and toxin-induced inflammatory demyelinating conditions in the brain. Furthermore, the animal studies have yielded mechanistic insights into the molecular mechanisms of KD action in relevant situations, paving the way for precision nutrition. Herein we review and synthesize recent advances and also identify unresolved issues, such as the roles of adipokines and gut microbiota, in this field. Hopefully this panoramic view of current understanding can inform future research directions and clinical practice with regard to KD in MS and related conditions.

Androgen Receptor Enhances the Efficacy of Sorafenib Against Hepatocellular Carcinoma Through Enriched EpCAM Stemness.

BACKGROUND/AIM: The role of androgen receptor (AR) in hepatocellular carcinoma (HCC) development is controversial. Therefore, the translational value of targeting AR in HCC is unknown. Sorafenib, a multiple kinase inhibitor, is the standard therapy for patients with unresectable HCC. This study investigated sorafenib effect on AR in experimental models of HCC. MATERIAL AND METHODS: AR cDNA was introduced into HCC cells and in vitro cell growth and in vivo tumor growth were measured. Sphere cells, as well as epithelial cell adhesion molecule-positive (EpCAM+) and CD133+ cells were isolated from HCC cells with/without AR expression to observe in vitro/in vivo effects. Liver specific AR knockout in mouse models of spontaneous HCC (carcinogen-induced and hepatitis B virus-related HCC) was also implemented to examine gene expression. HCC cells/tumors were treated with sorafenib in order to determine effects on tumor growth and related gene expression. RESULT: AR cDNA increased transactivation function, increased colony/sphere-forming activities, and enhanced tumorigenicity in HCC cells compared to their parental cells. Expression of the stemness marker EpCAM was also dramatically increased. In carcinogen-and HBV-induced HCC models, EpCAM+ cells were significantly reduced in AR-knockout mice compared to wild-type HCCs. In addition, AR reduced sorafenib-related signals, e.g. extracellular-regulated kinase, AKT serine/threonine kinase 1, and p38 mitogen-activated protein kinase, compared to that in parental cells. Regarding sorafenib cytotoxicity, AR-expressing cells were vulnerable to treatment. Moreover, the half maximal-inhibitory concentration (IC50) was drastically lowered in AR+/EpCAM+ compared to AR-/EpCAM- sphere cells. Strikingly, the IC50 in AR+/CD133+ vs. AR-/CD133+ cells were similar. Moreover, sorafenib robustly suppressed tumor growth in implanted AR+/EpCAM+ cells but not AR-/EpCAM- ones. Finally, bioinformatics analyses revealed EpCAM to be a prognostic biomarker in Asian and non-alcohol-consuming patients with HCC, suggesting suitability of a sorafenib regimen for such patients. CONCLUSION: AR+/EpCAM+ may be a marker of responsiveness to sorafenib for patients with HCC. Prospective surveys associating AR/EpCAM expression with therapy outcomes are essential.

Survival benefit of sphingosin-1-phosphate and receptors expressions in breast cancer patients.

Sphingosine-1-phosphate (S1P) is a bioactive lipid that exerts various pathophysiological functions through binding to its receptor family (S1PRs). Since first report of the breast cancer (BCA) promoting function by S1P production (through the function of sphingosine kinases) and S1P/S1PR signaling, their antagonists have never been successfully progress to clinics after three decades. Taking advantage of bioinformatics linking to gene expression to disease prognosis, we examined the impact of associated genes in BCA patients. We found high gene expressions involved in S1P anabolism suppressed disease progression of patients who are basal cell type BCA or receiving adjuvant therapy. In addition, S1PRs expression also suppressed disease progress of multiple categories of BCA patient progression. This result is contradictory to tumor promoter role of S1P/S1PRs which revealed in the literature. Further examination by directly adding S1P in BCA cells found a cell growth suppression function, which act via the expression of S1PR1. In conclusion, our study is the first evidence claiming a survival benefit function of S1P/S1PR signaling in BCA patients, which might explain the obstacle of relative antagonist apply in clinics.

Androgen receptor enhances cell adhesion and decreases cell migration via modulating ß1-integrin-AKT signaling in hepatocellular carcinoma cells.

The androgen receptor (AR) has been shown to promote the initiation and development of hepatocellular carcinoma (HCC) during the early stage of the disease process and to suppress HCC cell invasion during the later stages of the disease. The mechanisms governing these dual yet opposite roles have yet to be elucidated. Using carcinogen-induced HCC in vivo mouse models and the in vitro human HCC cell line SKhep1, we found that knockout of AR in primary HCC cells led to a decrease in HCC cell focal adhesion capacity compared to cells from wildtype mice. Similar results were obtained after adding functional AR into human HCC SKhep1 cells. Further analysis revealed that the role AR plays in adhesion of HCC cells is governed, at least in part, by its ability to up-regulate ß1-integrin and activate the PI3K/AKT pathway. We also found that AR-ß1-integrin-mediated cell adhesion suppresses cell migration. Those findings indicate that the AR-ß1-integrin-PI3K/AKT signaling pathway might play a role in the bimodal function of AR on cell adhesion and migration at the cellular level.

Glycogen synthase kinase 3 alpha and 3 beta have distinct functions during cardiogenesis of zebrafish embryo.

BACKGROUND: Glycogen synthase kinase 3 (GSK3) encodes a serine/threonine protein kinase, is known to play roles in many biological processes. Two closely related GSK3 isoforms encoded by distinct genes: GSK3alpha (51 kDa) and GSK3beta (47 kDa). In previously studies, most GSK3 inhibitors are not only inhibiting GSK3, but are also affecting many other kinases. In addition, because of highly similarity in amino acid sequence between GSK3alpha and GSK3beta, making it difficult to identify an inhibitor that can be selective against GSK3alpha or GSK3beta. Thus, it is relatively difficult to address the functions of GSK3 isoforms during embryogenesis. At this study, we attempt to specifically inhibit either GSK3alpha or GSK3beta and uncover the isoform-specific roles that GSK3 plays during cardiogenesis. RESULTS: We blocked gsk3alpha and gsk3beta translations by injection of morpholino antisense oligonucleotides (MO). Both gsk3alpha- and gsk3beta-MO-injected embryos displayed similar morphological defects, with a thin, string-like shaped heart and pericardial edema at 72 hours post-fertilization. However, when detailed analysis of the gsk3alpha- and gsk3beta-MO-induced heart defects, we found that the reduced number of cardiomyocytes in gsk3alpha morphants during the heart-ring stage was due to apoptosis. On the contrary, gsk3beta morphants did not exhibit significant apoptosis in the cardiomyocytes, and the heart developed normally during the heart-ring stage. Later, however, the heart positioning was severely disrupted in gsk3beta morphants. bmp4 expression in gsk3beta morphants was up-regulated and disrupted the asymmetry pattern in the heart. The cardiac valve defects in gsk3beta morphants were similar to those observed in axin1 and apcmcr mutants, suggesting that GSK3beta might play a role in cardiac valve development through the Wnt/beta-catenin pathway. Finally, the phenotypes of gsk3alpha mutant embryos cannot be rescued by gsk3beta mRNA, and vice versa, demonstrating that GSK3alpha and GSK3beta are not functionally redundant. CONCLUSION: We conclude that (1) GSK3alpha, but not GSK3beta, is necessary in cardiomyocyte survival; (2) the GSK3beta plays important roles in modulating the left-right asymmetry and affecting heart positioning; and (3) GSK3alpha and GSK3beta play distinct roles during zebrafish cardiogenesis.

Specific stabilization of DNA triple helices by indolo[2,1-b]quinazolin-6,12-dione derivatives.

Derivatives of indolo[2,1-b]quinazolinone containing aminoalkylamino side chains were synthesized as specific DNA triplex stabilizing agents. The aminoalkylamino side chains are essential for triplex stabilization. The position-8 fluorine atom or a methyl group to the nitrogen adjacent to the planar core can enhance triplex stability by 6 degrees C and the effect is additive. Conformational analysis reveals that the orientation of the side chain underlies the ability of this compound to stabilize a DNA triplex.