Changes in the Acetylcholinesterase Enzymatic Activity in Tumor Development and Progression.

Acetylcholinesterase is a well-known protein because of the relevance of its enzymatic activity in the hydrolysis of acetylcholine in nerve transmission. In addition to the catalytic action, it exerts non-catalytic functions; one is associated with apoptosis, in which acetylcholinesterase could significantly impact the survival and aggressiveness observed in cancer. The participation of AChE as part of the apoptosome could explain the role in tumors, since a lower AChE content would increase cell survival due to poor apoptosome assembly. Likewise, the high Ach content caused by the reduction in enzymatic activity could induce cell survival mediated by the overactivation of acetylcholine receptors (AChR) that activate anti-apoptotic pathways. On the other hand, in tumors in which high enzymatic activity has been observed, AChE could be playing a different role in the aggressiveness of cancer; in this review, we propose that AChE could have a pro-inflammatory role, since the high enzyme content would cause a decrease in ACh, which has also been shown to have anti-inflammatory properties, as discussed in this review. In this review, we analyze the changes that the enzyme could display in different tumors and consider the different levels of regulation that the acetylcholinesterase undergoes in the control of epigenetic changes in the mRNA expression and changes in the enzymatic activity and its molecular forms. We focused on explaining the relationship between acetylcholinesterase expression and its activity in the biology of various tumors. We present up-to-date knowledge regarding this fascinating enzyme that is positioned as a remarkable target for cancer treatment.

Erk1/2 signaling mediates the HGF-induced protection against ethanol and acetaldehyde-induced toxicity in the pancreatic RINm5F cell line.

Alcohol-induced pancreas damage remains as one of the main risk factors for pancreatitis development. This disorder is poorly understood, particularly the effect of acetaldehyde, the primary alcohol metabolite, in the endocrine pancreas. Hepatocyte growth factor (HGF) is a protective protein in many tissues, displaying antioxidant, antiapoptotic, and proliferative responses. In the present work, we were focused on characterizing the response induced by HGF and its protective mechanism in the RINm5F pancreatic cell line treated with ethanol and acetaldehyde. RINm5F cells were treated with ethanol or acetaldehyde for 12 h in the presence or not of HGF (50 ng/ml). Cells under HGF treatment decreased the content of reactive oxygen species and lipid peroxidation induced by both toxics, improving cell viability. This effect was correlated to an improvement in insulin expression impaired by ethanol and acetaldehyde. Using a specific inhibitor of Erk1/2 abrogated the effects elicited by the growth factor. In conclusion, the work provides mechanistic evidence of the HGF-induced-protective response to the alcohol-induced damage in the main cellular component of the endocrine pancreas.

A low-intensity lifelong exercise routine changes miRNA expression in aging and prevents osteosarcopenic obesity by modulating inflammation.

Osteosarcopenic obesity (OSO) has been associated with increase immobility, falls, fractures, and other dysfunctions, which could increase mortality risk during aging. However, its etiology remains unknown. Recent studies revealed that sedentarism, fat gain, and epigenetic regulators are critical in its development. One effective intervention to prevent and treat OSO is exercise. Therefore, in the present study, by keeping rats in conditions of sedentarism and others under a low-intensity exercise routine, we established an experimental model of OSO. We determined the degree of sarcopenia, obesity, and osteopenia at different ages and analyzed the miRNA expression during the lifespan using miRNA microarrays from gastrocnemius muscle. Interestingly microarrays results showed that there is a set of miRNAs that changed their expression with exercise. The pathway enrichment analysis showed that these miRNAs are strongly associated with immune regulation. Further inflammatory profiles with IL-6/IL-10 and TNF-α/IL-10 ratios showed that exercised rats presented a lower pro-inflammatory profile than sedentary rats. Also, the body fat gain in the sedentary group increased the inflammatory profile, ultimately leading to muscle dysfunction. Exercise prevented strength loss over time and maintained skeletal muscle functionality over time. Differential expression of miRNAs suggests that they might participate in this process by regulating the inflammatory response associated with aging, thus preventing the development of OSO.

The hepatic effects of GDF11 on health and disease.

The growth differentiation factor 11 (GDF11), a member of the superfamily of the transforming growth factor ß, has gained relevance in the last few years due to its remarkable effects in cellular biology, particularly in the nervous system, skeletal muscle, the heart, and many epithelial tissues. Some controversies have been raised about this growth factor. Many of them have been related to technical factors but also the nature of the cellular target. In liver biology and pathobiology, the GDF11 has shown to be related in many molecular aspects, with a significant impact on the physiology and the initiation and progression of the natural history of liver diseases. GDF11 has been involved as a critical regulator in lipid homeostasis, which, as it is well known, is the first step in the progression of liver disease. However, also it has been reported that the GDF11 is involved in fibrosis, senescence, and cancer. Although there are some controversies, much of the literature indicates that GDF11 displays effects tending to solve or mitigate pathological states of the liver, with reasonable evidence of correlation with other organs or systems. To a large extent, the controversy, as mentioned, is due to technical problems, such as the specificity of GDF11 antibodies, confusion with its closer family member, myostatin, and the state of differentiation in the tissues. In the present work, we reviewed the specific effects of GDF11 in the biology and pathobiology of the liver as a potential and promising factor for therapeutic intervention shortly.

Folate Metabolism in Hepatocellular Carcinoma. What Do We Know So Far?

Cancer cells are characterized by accelerated proliferation and an outstanding adaptation of their metabolic pathways to meet energy demands. The folate cycle, also known as folate metabolism or one-carbon metabolism, through enzymatic interconversions, provides metabolites necessary for nucleotide synthesis, methylation, and reduction power, helping to maintain the high rate of proliferation; therefore, the study of this metabolic pathway is of great importance in the study of cancer. Moreover, multiple enzymes involved in this cycle have been implicated in different types of cancer, corroborating the cell's adaptations under this pathology. During the last decade, nonalcoholic fatty liver disease has emerged as the leading etiology related to the rise in the incidence and deaths of hepatocellular carcinoma. Specifically, cholesterol accumulation has been a determinant promoter of tumor formation, with solid evidence that an enriched-cholesterol diet plays a crucial role in accelerating the development of an aggressive subtype of hepatocellular carcinoma compared to other models. In this review, we will discuss the most recent findings to understand the contribution of folate metabolism to cancer cells and tumor microenvironment while creating a link between the dynamics given by cholesterol and methylenetetrahydrofolate dehydrogenase 1-like, a key enzyme of the cycle located in the mitochondrial compartment.

The mechanism of the cadmium-induced toxicity and cellular response in the liver.

Cadmium is a toxic element to which man can be exposed at work or in the environment. Cd's most salient toxicological property is its exceptionally long half-life in the human body. Once absorbed, Cd accumulates in the human body, particularly in the liver. The cellular actions of Cd are extensively documented, but the molecular mechanisms underlying these actions are still not resolved. The liver manages the cadmium to eliminate it by a diverse mechanism of action. Still, many cellular and physiological responses are executed in the task, leading to worse liver damage, ranging from steatosis, steatohepatitis, and eventually hepatocellular carcinoma. The progression of cadmium-induced liver damage is complex, and it is well-known the cellular response that depends on the time in which the metal is present, ranging from oxidative stress, apoptosis, adipogenesis, and failures in autophagy. In the present work, we aim to present a review of the current knowledge of cadmium toxicity and the cellular response in the liver.

The Critical Role of Hypoxia in the Re-Differentiation of Human Articular Chondrocytes.

The preservation of the chondrogenic phenotype and hypoxia-related physiological microenvironment are major challenges in the 2D culture of primary human chondrocytes. To address this problem, we develop a 3D culture system generating scaffold-free spheroids from human chondrocytes. Our results highlight the chondrogenic potential of cultured human articular chondrocytes in a 3D system combined with hypoxia independently of the cartilage source. After 14 days of culture, we developed spheroids with homogenous diameter and shape from hyaline cartilage donors. Spheroids generated in hypoxia showed a significantly increased glycosaminoglycans synthesis and up-regulated the expression of SOX9, ACAN, COL2A1, COMP, and SNAI1 compared to those obtained under normoxic conditions. Therefore, we conclude that spheroids developed under hypoxic conditions modulate the expression of chondrogenesis-related genes and native tissue features better than 2D cultures. Thus, this scaffold-free 3D culture system represents a novel in vitro model that can be used for cartilage biology research.

Hepatocyte growth factor reverses cholemic nephropathy associated with α-naphthylisothiocyanate-induced cholestasis in mice.

Hepatocyte growth factor (HGF) has been proved to protect the liver against α-naphthylisothiocyanate (ANIT)-induced cholestasis by acting as an antioxidant agent and redirecting toxic biliary solutes towards blood for urinary excretion. However, this may represent an additional potential risk for kidney integrity, which is already compromised by the cholestatic process itself (cholemic nephropathy). Therefore, in the present work, we studied the renal damage caused by ANIT-induced cholestasis and whether it is aggravated or, on the contrary, counteracted by HGF; if the latter holds, the involvement of its antioxidant properties will be ascertained. ANIT-induced cholestatic deleterious renal effects were corroborated by the presence of urine bile salts, impairment of renal function, and the alterations of renal damage markers, such as HSP72, creatinine clearance, and albuminuria. HGF fully reverted all these, and the cast formation in the tubules was significantly decreased. These findings were associated with the control of renal oxidative stress. In summary, despite HGF enhancing the overload of potentially harmful biliary constituents that the kidney should remove from the bloodstream as an alternative depuration organ in cholestasis, it simultaneously protects the kidney from this damage by counteracting the prooxidant effects resulting from this harmful exposure.

Influence of genetic and environmental risk factors in the development of hepatocellular carcinoma in Mexico.

The latest studies on the epidemiology of diverse types of cancers have located in the scene the relevance of liver tumors, particularly hepatocellular carcinoma (HCC). HCC is a life-threatening malignancy triggered by chronic exposure to hepatitis B and C viruses, excessive alcohol intake, hepatic lipid droplet accumulation, and aflatoxins that lead to persistent liver damage. The occurrence of such etiological risk factors deeply marks the variability in the incidence of HCC worldwide reflected by geography, ethnicity, age, and lifestyle factors influenced by cultural aspects. New perspectives on the primary risk factors and their potential gene-environment interactions (GxE) have been well-addressed in some cancers; however, it continues to be a partially characterized issue in liver malignancies. In this review, the epidemiology of the risk factors for HCC are described enhancing the GxE interactions identified in Mexico, which could mark the risk of this liver malignancy among the population and the measures needed to revert them. Updated healthcare policies focusing on preventive care should be tailored based on the genetic and environmental risk factors, which may influence the effect of the etiological agents of HCC. Robust regional investigations related to epidemiological, clinical, and basic studies are warranted to understand this health problem complying with the rules of ethnic, genetic, environmental, and social diversity.

GLUT4 translocation in C2C12 myoblasts and primary mouse hepatocytes by an antihyperglycemic flavone from Tillandsia usneoides.

BACKGROUND: Type 2 Diabetes (T2D) is characterized by deregulation in carbohydrate and lipid metabolism, with a very high mortality rate. Glucose Transporter type 4 (GLUT4) plays a crucial role in T2D and represents a therapeutic target of interest. Tillandsia usneoides (T. usneoides) is a plant used as a remedy for diabetes. T. usneoides decreased blood glucose in different experimental models. However, the involvement of GLUT4 in this effect has not yet been explored. PURPOSE: This study aimed to investigate whether any component in T. usneoides might participate in the effect on blood glucose through a bioassay-guided fractionation, testing its potential antihyperglycemic effect in mice, as well as its influence on GLUT4 translocation in C2C12 myoblasts and primary hepatocytes. METHODS: The aqueous extract and the Ethyl Acetate fraction (TU-AcOEt) of T. usneoides were evaluated in a hypoglycemic activity bioassay and in the glucose tolerance test in CD-1 mice. TU-AcOEt was fractionated, obtaining five fractions that were studied in an additional glucose tolerance test. C1F3 was fractioned again, and its fractions (C2F9-12, C2F22-25, and C2F38-44) were examined by HPLC. The C2F38-44 fraction was analyzed by Mass Spectrometry (MS) and subjected to additional fractionation. The fraction C3F6-9 was explored by Nuclear Magnetic Resonance (NMR), resulting in 5,7,4´-trihydroxy-3,6,3´,5´-tetramethoxyflavone (Flav1). Subsequently, a viability test was performed to evaluate the cytotoxic effect of Flav1 and fractions C2F9-12, C2F22-25. C2F38-44, and C3F30-41 in C2C12 myoblasts and primary mouse hepatocytes. Confocal microscopy was also performed to assess the effect of Flav1 and fractions on GLUT4 translocation. RESULTS: The TU-AcOEt fraction exhibited a hypoglycemic and antihyperglycemic effect in mice, and its fractionation resulted in five fractions, among which fraction C1F3 decreased blood glucose. MS and NMR analysis revealed the presence of Flav1. Finally, Flav1 significantly promoted the translocation of GLUT4 in C2C12 myoblasts and primary hepatocytes. CONCLUSION: To date, Flav1 has not been reported to have activity in GLUT4; this study provides evidence that T. usneoides is a plant with the potential to develop novel therapeutic agents for the control of T2D.

The Consumption of Cholesterol-Enriched Diets Conditions the Development of a Subtype of HCC with High Aggressiveness and Poor Prognosis.

Non-alcoholic fatty liver disease (NAFLD) and progression to non-alcoholic steatohepatitis (NASH) result as a consequence of diverse conditions, mainly unbalanced diets. Particularly, high-fat and cholesterol content, as well as carbohydrates, such as those commonly ingested in Western countries, frequently drive adverse metabolic alterations in the liver and promote NAFLD development. Lipid liver overload is also one of the main risk factors for initiation and progression of hepatocellular carcinoma (HCC), but detailed knowledge on the relevance of high nutritional cholesterol remains elusive. We were aimed to characterize HCC development in mice fed with a Western diet (high in lipids and cholesterol) and to identify molecular alterations that define a subtype of liver cancer induced by lipid overload. Mice under western or high cholesterol diets more frequently developed tumors with a more aggressive phenotype than animals fed with a chow diet. Associated changes involved macrophage infiltration, angiogenesis, and stemness features. RNA-seq revealed a specific gene expression signature (Slc41a; Fabp5; Igdcc4 and Mthfd1l) resembling the adverse phenotypic features and poor clinical outcomes seen in patients with HCC. In conclusion; consumption of lipid enriched diets; particularly cholesterol; could accelerate HCC development with an aggressive phenotype and poor prognosis.

HGF/c-Met regulates p22phox subunit of the NADPH oxidase complex in primary mouse hepatocytes by transcriptional and post-translational mechanisms.

INTRODUCTION AND OBJECTIVES: It is well-known that signaling mediated by the hepatocyte growth factor (HGF) and its receptor c-Met in the liver is involved in the control of cellular redox status and oxidative stress, particularly through its ability to induce hepatoprotective gene expression by activating survival pathways in hepatocytes. It has been reported that HGF can regulate the expression of some members of the NADPH oxidase family in liver cells, particularly the catalytic subunits and p22phox. In the present work we were focused to characterize the mechanism of regulation of p22phox by HGF and its receptor c-Met in primary mouse hepatocytes as a key determinant for cellular redox regulation. MATERIALS AND METHODS: Primary mouse hepatocytes were treated with HGF (50 ng/mL) at different times. cyba expression (gene encoding p22phox) or protein content were addressed by real time RT-PCR, Western blot or immunofluorescence. Protein interactions were explored by immunoprecipitation and FRET analysis. RESULTS: Our results provided mechanistic information supporting the transcriptional repression of cyba induced by HGF in a mechanism dependent of NF-κB activity. We identified a post-translational regulation mechanism directed by p22phox degradation by proteasome 26S, and a second mechanism mediated by p22phox sequestration by c-Met in plasma membrane. CONCLUSION: Our data clearly show that HGF/c-Met exerts regulation of the NADPH oxidase by a wide-range of molecular mechanisms. NADPH oxidase-derived reactive oxygen species regulated by HGF/c-Met represents one of the main mechanisms of signal transduction elicited by this growth factor.

GDF11 restricts aberrant lipogenesis and changes in mitochondrial structure and function in human hepatocellular carcinoma cells.

Growth differentiation factor 11 (GDF11) has been characterized as a key regulator of differentiation in cells that retain stemness features. Recently, it has been reported that GDF11 exerts tumor-suppressive properties in hepatocellular carcinoma cells, decreasing clonogenicity, proliferation, spheroid formation, and cellular function, all associated with a decrement in stemness features, resulting in mesenchymal to epithelial transition and loss of aggressiveness. The aim of the present work was to investigate the mechanism associated with the tumor-suppressive properties displayed by GDF11 in liver cancer cells. Hepatocellular carcinoma-derived cell lines were exposed to GDF11 (50 ng/ml), RNA-seq analysis in Huh7 cell line revealed that GDF11 exerted profound transcriptomic impact, which involved regulation of cholesterol metabolic process, steroid metabolic process as well as key signaling pathways, resembling endoplasmic reticulum-related functions. Cholesterol and triglycerides determination in Huh7 and Hep3B cells treated with GDF11 exhibited a significant decrement in the content of these lipids. The mTOR signaling pathway was downregulated, and this was associated with a reduction in key proteins involved in the mevalonate pathway. In addition, real-time metabolism assessed by Seahorse technology showed abridged glycolysis as well as glycolytic capacity, closely related to an impaired oxygen consumption rate and decrement in adenosine triphosphate production. Finally, transmission electron microscopy revealed mitochondrial abnormalities, such as cristae disarrangement, consistent with metabolic changes. Results provide evidence that GDF11 impairs cancer cell metabolism targeting lipid homeostasis, glycolysis, and mitochondria function and morphology.

Mediterranean-like mix of fatty acids induces cellular protection on lipid-overloaded hepatocytes from western diet fed mice.

INTRODUCTION AND OBJECTIVE: Non-alcoholic fatty liver disease remains as one of the main liver disorders worldwide. It is widely accepted that is the kind of lipid, rather than the amount deposited in the cells that determines cell damage. Cholesterol and saturated free fatty acids are deleterious lipids when accumulated but, in contrast, there are some valuable lipids that could counteract those with harmful properties. Much of this knowledge arises from studies using a single fatty acid, but the effects of a combination of fatty acids, as obtained by diet has been poorly addressed. In the present work, we were focused to figure out the cellular effect of two different mixes of fatty acids, one with high proportion of saturated fatty acids, and another one with high proportion of unsaturated fatty acids (Mediterranean-like) in a cellular model of steatosis. MATERIAL AND METHODS: Primary mouse hepatocytes from animals fed with a western diet (high fat and carbohydrates diet), were treated with both mixes of fatty acids for 24 h. RESULTS: Our data clearly show that only the high unsaturated fatty acid mix induced a decrease in triglycerides (47.5%) and cholesterol (59%) content in steatotic hepatocytes mediating cellular protection associated to the decrement of ROS and oxidative damage. The mixture of high saturated fatty acids exhibited no effects, preserving high levels of cholesterol and triglycerides and oxidative damage. In conclusion, our results show that Mediterranean-like mix of fatty acids exerts cellular protection in steatosis by decreasing triglycerides, cholesterol, ROS content and oxidative damage.

Regulation of Cas9 by viral proteins Tat and Rev for HIV-1 inactivation.

While combined antiretroviral therapy (cART) has had a great impact on the treatment of HIV-1 infection, the persistence of long-lived cells with an intact provirus precludes virus eradication and sterilizing cure. CRISPR/Cas9 genome editing has become an efficient tool to eradicate HIV-1 genome or prevent replication. Furthermore, regulation of Cas9 gene expression by HIV can induce mutations that could inactivate the proviral genome, making a gene therapy safe by preventing the induction of non-specific mutations, which could compromise the integrity of healthy cells. In this study, isolated HIV-1 LTR, INS and RRE sequences were used to regulate Cas9 expression in HEK293 cells, and guide RNAs (gRNAs) were designed to target mutations in HIV-1 conserved regions such as tat and rev regulatory genes. We demonstrate that Cas9 expression in our system is controlled by the HIV-1 Tat and Rev proteins, leading to self-regulation of gene edition, and showing a strong antiviral effect by inactivating HIV-1 replication. Sequencing analysis confirmed that viral genome was partially excised by multiplex editing (90% efficiency), and viral capsid protein (CA-p24) was undetectable. In conclusion, the self-regulated CRISPR/Cas9 system may be a reliable and accurate strategy for eliminating HIV-1 infection whose effect will be restricted to infected cells.

GDF11 Implications in Cancer Biology and Metabolism. Facts and Controversies.

Growth Differentiation Factor 11 (GDF11), a member of the super family of the Transforming Growth Factor ß, has gained more attention in the last few years due to numerous reports regarding its functions in other systems, which are different to those related to differentiation and embryonic development, such as age-related muscle dysfunction, skin biology, metabolism, and cancer. GDF11 is expressed in many tissues, including skeletal muscle, pancreas, kidney, nervous system, and retina, among others. GDF11 circulating levels and protein content in tissues are quite variable and are affected by pathological conditions or age. Although, GDF11 biology had a lot of controversies, must of them are only misunderstandings regarding the variability of its responses, which are independent of the tissue, grade of cellular differentiation or pathologies. A blunt fact regarding GDF11 biology is that its target cells have stemness feature, a property that could be found in certain adult cells in health and in disease, such as cancer cells. This review is focused to present and analyze the recent findings in the emerging research field of GDF11 function in cancer and metabolism, and discusses the controversies surrounding the biology of this atypical growth factor.

GDF11 exhibits tumor suppressive properties in hepatocellular carcinoma cells by restricting clonal expansion and invasion.

Growth differentiation factor 11 (GDF11) has been characterized as a key regulator of differentiation in cells that retain stemness features, despite some controversies in age-related studies. GDF11 has been poorly investigated in cancer, particularly in those with stemness capacity, such as hepatocellular carcinoma (HCC), one of the most aggressive cancers worldwide. Here, we focused on investigating the effects of GDF11 in liver cancer cells. GDF11 treatment significantly reduced proliferation, colony and spheroid formation in HCC cell lines. Consistently, down-regulation of CDK6, cyclin D1, cyclin A, and concomitant upregulation of p27 was observed after 24 h of treatment. Interestingly, cell viability was unchanged, but cell functionality was compromised. These effects were potentially induced by the expression of E-cadherin and occludin, as well as Snail and N-cadherin repression, in a time-dependent manner. Furthermore, GDF11 treatment for 72 h induced that cells were incapable of sustaining colony and sphere capacity in the absent of GDF11, up to 5 days, indicating that the effect of GDF11 on self-renewal capacity is not transient. Finally, in vivo invasion studies revealed a significant decrease in cell migration of hepatocellular carcinoma cells treated with GDF11 associated to a decreased proliferation judged by Ki67 staining. Data show that exogenous GDF11 displays tumor suppressor properties in HCC cells.

Cholangiocyte death in ductopenic cholestatic cholangiopathies: Mechanistic basis and emerging therapeutic strategies.

Among hepatic diseases, cholestatic ductopenic cholangiopathies are poorly studied, and they are rarely given the importance they deserve, especially considering their high incidence in clinical practice. Although cholestatic ductopenic cholangiopathies have different etiologies and pathogenesis, all have the same target (the cholangiocyte) and similar mechanistic basis of cell death. Cholestatic cholangiopathies are characterized, predominantly, by obstructive or functional damage in the biliary epithelium, resulting in an imbalance between proliferation and cholangiocellular death; this leads to the progressive disappearance of bile ducts, as has been shown to occur in primary sclerosing cholangitis, primary biliary cholangitis, low-phospholipid-associated cholelithiasis syndrome, cystic fibrosis-related liver disease, and drug-induced ductopenia, among other biliary disorders. This review summarizes the features of the more common ductopenic syndromes and the cellular mechanisms involved in cholengiocellular death, with focus on the main forms of cholangiocyte death described so far, namely apoptosis, autophagy, necrosis, and necroptosis. It also emphasizes the importance to study in depth the molecular mechanisms of cholengiocyte death to make possible to counteract them with therapeutic purposes. These therapeutic strategies are limited in number and efficacy at present, and this is why it is important to find complementary, safe strategies to stimulate cholangiocellular proliferation in order favor bile duct replenishment as well. Successful in finding appropriate treatments would prevent the patient from having liver transplantation as the only therapeutic alternative.

Cholesterol burden in the liver induces mitochondrial dynamic changes and resistance to apoptosis.

Non-alcoholic fatty liver disease (NAFLD) encompasses a broad spectrum of histopathological changes ranging from non-inflammatory intracellular fat deposition to non-alcoholic steatohepatitis (NASH), which may progress into hepatic fibrosis, cirrhosis, or hepatocellular carcinoma. Recent data suggest that impaired hepatic cholesterol homeostasis and its accumulation are relevant to the pathogenesis of NAFLD/NASH. Despite a vital physiological function of cholesterol, mitochondrial dysfunction is an important consequence of dietary-induced hypercholesterolemia and was, subsequently, linked to many pathophysiological conditions. The aim in the current study was to evaluate the morphological and molecular changes of cholesterol overload in mouse liver and particularly, in mitochondria, induced by a high-cholesterol (HC) diet for one month. Histopathological studies revealed microvesicular hepatic steatosis and significantly elevated levels of liver cholesterol and triglycerides leading to impaired liver synthesis. Further, high levels of oxidative stress could be determined in liver tissue as well as primary hepatocyte culture. Transcriptomic changes induced by the HC diet involved disruption in key pathways related to cell death and oxidative stress as well as upregulation of genes related to glutathione homeostasis. Impaired liver function could be associated with a decrease in mitochondrial membrane potential and ATP content and significant alterations in mitochondrial dynamics. We demonstrate that cholesterol overload in the liver leads to mitochondrial changes which may render damaged hepatocytes proliferative and resistant to cell death whereby perpetuating liver damage.

Ginkgo biloba induces different gene expression signatures and oncogenic pathways in malignant and non-malignant cells of the liver.

Ginkgo biloba (EGb761) is a widely used botanical drug. Several reports indicate that EGb761 confers preventive as well as anti-tumorigenic properties in a variety of tumors, including hepatocellular carcinoma (HCC). We here evaluate functional effects and molecular alterations induced by EGb761 in hepatoma cells and non-malignant hepatocytes. Hepatoma cell lines, primary human HCC cells and immortalized human hepatocytes (IH) were exposed to various concentrations (0-1000 µg/ml) of EGb761. Apoptosis and proliferation were evaluated after 72h of EGb761 exposure. Response to oxidative stress, tumorigenic properties and molecular changes were further investigated. While anti-oxidant effects were detected in all cell lines, EGb761 promoted anti-proliferative and pro-apoptotic effects mainly in hepatoma cells. Consistently, EGb761 treatment caused a significant reduction in colony and sphere forming ability in hepatoma cells and no mentionable changes in IH. Transcriptomic changes involved oxidative stress response as well as key oncogenic pathways resembling Nrf2- and mTOR signaling pathway. Taken together, EGb761 induces differential effects in non-transformed and cancer cells. While treatment confers protective effects in non-malignant cells, EGb761 significantly impairs tumorigenic properties in cancer cells by affecting key oncogenic pathways. Results provide the rational for clinical testing of EGb761 in preventive and therapeutic strategies in human liver diseases.