Bitter melon extract suppresses metastatic breast cancer cells (MCF-7 cells) growth possibly by hindering glucose uptake.

Breast cancer is one of the most commonly diagnosed cancers among women, however the complete cure for metastatic breast cancer is lacking due to poor prognosis. There has been an increasing trend of dietary modifications including consumption of natural food for the prevention of cancer. One of the popular natural foods is bitter melon. Bitter melon grows in tropical and subtropical areas. Some of the beneficial effects of bitter melon towards disease including cancer have been reported at the whole body/organismal level. However, specific cellular mechanisms by which bitter melon exerts beneficial effects in breast cancer are lacking. In this study, we used a human metastatic breast cancer cell line, MCF-7 cell, to study if bitter melon alters glucose clearance from the culture medium. We co-cultured MCF-7 cells with bitter melon extract in the presence and absence of supplemented insulin and subsequently measured MCF-7 cells viability. In this study, we report a noble finding that bitter melon extract exerts cytotoxic effects on MCF-7 cells possibly via inhibition of glucose uptake. Our findings show that insulin rescues MCF-7 cells from the effects of bitter melon extract.

NRF2 transcriptionally regulates Caspase-11 expression to activate HMGB1 release by Autophagy-deficient hepatocytes.

Injury or stress can induce intracellular translocation and release of nuclear HMGB1, a DAMP molecule known to participate in inflammation and other pathological processes. Active release of HMGB1 from stimulated macrophages can be mediated by inflammasomes, which cleave Gasdermin D to form pores on cytoplasmic membranes. We previously had shown that active release of HMGB1 from autophagy deficient hepatocytes also depended on the inflammasome but how the inflammasome was activated was not known. Here we report that persistent activation of transcription factor NRF2 under the autophagy deficient condition led to transcriptional upregulation of Caspase-11 expression, which could then activate the CASPASE-1inflammasome. Using chromatin immunoprecipitation (CHIP) and luciferase-based reporter assays, we show that NRF2 directly binds to the Caspase-11 promoter and transcriptionally increase the expression of Caspase-11. Genetic deletion of Caspase-11 in autophagy-deficient livers represses the release of HMGB1 and its pathological consequence, ductular cell proliferation. Consistently, deletion of NLRP3, which can activate CASPASE-1 mediated inflammasomes under other types of signals, did not prevent HMGB1 release and ductular cell proliferation in autophagy deficient livers. Surprisingly, while cleavage of GASDEMIN D occurred in autophagy-deficient livers its deletion did not prevent the HMGB1 release, suggesting that CASPASE-11-mediated inflammasome activation may also engage in a different mechanism for HMGB1 release by the autophagy deficient hepatocytes. Collectively, this work reveals the novel role of NRF2 in transcriptional upregulation of Caspase-11 and in inflammasome activation to promote active release of HMGB via a non-Gasdermin D mediated avenue.

Impaired hepatic autophagy exacerbates hepatotoxin induced liver injury.

Hepatotoxins activate the hepatic survival pathway, but it is unclear whether impaired survival pathways contribute to liver injury caused by hepatotoxins. We investigated the role of hepatic autophagy, a cellular survival pathway, in cholestatic liver injury driven by a hepatotoxin. Here we demonstrate that hepatotoxin contained DDC diet impaired autophagic flux, resulting in the accumulation of p62-Ub-intrahyaline bodies (IHBs) but not the Mallory Denk-Bodies (MDBs). An impaired autophagic flux was associated with a deregulated hepatic protein-chaperonin system and significant decline in Rab family proteins. Additionally, p62-Ub-IHB accumulation activated the NRF2 pathway rather than the proteostasis-related ER stress signaling pathway and suppressed the FXR nuclear receptor. Moreover, we demonstrate that heterozygous deletion of Atg7, a key autophagy gene, aggravated the IHB accumulation and cholestatic liver injury. Conclusion: Impaired autophagy exacerbates hepatotoxin-induced cholestatic liver injury. The promotion of autophagy may represent a new therapeutic approach for hepatotoxin-induced liver damage.

Promotion of diet-induced obesity and metabolic syndromes by BID is associated with gut microbiota.

A growing body of evidence has indicated an expanding functional network of B-cell lymphoma 2 (BCL-2) family proteins beyond regulation of cell death and survival. Here, we examined the role and mechanisms of BH3 interacting-domain death agonist (BID), a pro-death BCL-2 family member, in the development of diet-induced metabolic dysfunction. Mice deficient in bid (bid-/- ) were resistant to high-fat diet (HFD)-induced obesity, hepatic steatosis, and dyslipidemia with an increased insulin sensitivity. Indirect calorimetry analysis indicated that bid deficiency increased metabolic rate and decreased respiratory exchange ratio, suggesting a larger contribution of lipids to overall energy expenditure. While expression of several genes related to lipid accumulation was only increased in wild-type livers, metabolomics analysis revealed a consistent reduction in fatty acids but an increase in certain sugars and Krebs cycle intermediates in bid-/- livers. Gut microbiota (GM) analysis indicated that HFD induced gut dysbiosis with differential patterns in wild-type and in bid-/- mice. Notably, abrogation of GM by antibiotics during HFD feeding eliminated the beneficial effects against obesity and hepatic steatosis conferred by the bid deficiency. Conclusion: These results indicate that the protective role of bid-deficiency against diet-induced metabolic dysfunction interacts with the function of GM.

Senescence Connects Autophagy Deficiency to Inflammation and Tumor Progression in the Liver.

BACKGROUND & AIMS: Cellular senescence frequently is present in injured livers. The induction mechanism and the pathologic role are not always clear. We aimed to understand the dynamics of senescence induction and progression, and the mechanism responsible for the pathology using a mouse model that disables the essential process of autophagy. METHODS: Mice deficient in key autophagy genes Atg7 or Atg5 in the liver were used. Senescence was measured using established cellular and molecular signatures. The mechanistic roles of nuclear factor erythroid 2 (NRF2), forkhead box K1, and C-C motif chemokine receptor 2 (CCR2) were assessed using mouse genetic models. Liver functions, pathology, and tumor development were measured using biochemical and histologic approaches. RESULTS: Inducible deletion of Atg7 rapidly up-regulated cyclin-dependent kinase inhibitors independently of injury and induced senescence-associated ß-galactosidase activities and senescence-associated secretory phenotype (SASP). Sustained activation of NRF2 was the major factor causing senescence by mediating oxidative DNA damage and up-regulating C-C motif chemokine ligand 2, a key component of autophagy-related SASP, via the NRF2-forkhead box K1 axis. Senescence was responsible for hepatic inflammation through CCR2-mediated recruitment of CD11b+ monocytes and CD3+ T cells. The CCR2-mediated process in turn enhanced senescence and SASP by up-regulating cyclin-dependent kinase inhibitors and chemokines. Thus, senescence and inflammation can mutually augment each other, forming an amplification loop for both events. The CCR2-mediated process also modulated liver injury and tumor progression at the later stage of autophagy deficiency-related pathology. CONCLUSIONS: These results provide the insight that hepatic senescence can occur early in the disease process, triggers inflammation and is enhanced by inflammation, and has long-term effects on liver injury and tumor progression.

Therapeutic regulation of autophagy in hepatic metabolism.

Metabolic homeostasis requires dynamic catabolic and anabolic processes. Autophagy, an intracellular lysosomal degradative pathway, can rewire cellular metabolism linking catabolic to anabolic processes and thus sustain homeostasis. This is especially relevant in the liver, a key metabolic organ that governs body energy metabolism. Autophagy's role in hepatic energy regulation has just begun to emerge and autophagy seems to have a much broader impact than what has been appreciated in the field. Though classically known for selective or bulk degradation of cellular components or energy-dense macromolecules, emerging evidence indicates autophagy selectively regulates various signaling proteins to directly impact the expression levels of metabolic enzymes or their upstream regulators. Hence, we review three specific mechanisms by which autophagy can regulate metabolism: A) nutrient regeneration, B) quality control of organelles, and C) signaling protein regulation. The plasticity of the autophagic function is unraveling a new therapeutic approach. Thus, we will also discuss the potential translation of promising preclinical data on autophagy modulation into therapeutic strategies that can be used in the clinic to treat common metabolic disorders.

Hepatic Autophagy Deficiency Remodels Gut Microbiota for Adaptive Protection via FGF15-FGFR4 Signaling.

BACKGROUND & AIMS: The functions of the liver and the intestine are closely tied in both physiological and pathologic conditions. The gut microbiota (GM) often cause deleterious effects during hepatic pathogenesis. Autophagy is essential for liver homeostasis, but the impact of hepatic autophagy function on liver-gut interaction remains unknown. Here we investigated the effect of hepatic autophagy deficiency (Atg5Δhep) on GM and in turn the effect of GM on the liver pathology. METHODS: Fecal microbiota were analyzed by 16S sequencing. Antibiotics were used to modulate GM. Cholestyramine was used to reduce the enterohepatic bile acid (BA) level. The functional role of fibroblast growth factor 15 (FGF15) and ileal farnesoid X receptor (FXR) was examined in mice overexpressing FGF15 gene or in mice given a fibroblast growth factor receptor-4 (FGFR4) inhibitor. RESULTS: Atg5Δhep causes liver injury and alterations of intestinal BA composition, with a lower proportion of tauro-conjugated BAs and a higher proportion of unconjugated BAs. The composition of GM is significantly changed with an increase in BA-metabolizing bacteria, leading to an increased expression of ileal FGF15 driven by FXR that has a higher affinity to unconjugated BAs. Notably, antibiotics or cholestyramine treatment decreased FGF15 expression and exacerbated liver injury. Consistently, inhibition of FGF15 signaling in the liver enhances liver injury. CONCLUSIONS: Deficiency of autophagy function in the liver can affect intestinal environment, leading to gut dysbiosis. Surprisingly, such changes provide an adaptive protection against the liver injury through the FGF15-FGFR4 signaling. Antibiotics use in the condition of liver injury may thus have unexpected adverse consequences via the gut-liver axis.

The HMGB1-RAGE axis modulates the growth of autophagy-deficient hepatic tumors.

Autophagy is an intracellular lysosomal degradative pathway important for tumor surveillance. Autophagy deficiency can lead to tumorigenesis. Autophagy is also known to be important for the aggressive growth of tumors, yet the mechanism that sustains the growth of autophagy-deficient tumors is not unclear. We previously reported that progression of hepatic tumors developed in autophagy-deficient livers required high mobility group box 1 (HMGB1), which was released from autophagy-deficient hepatocytes. In this study we examined the pathological features of the hepatic tumors and the mechanism of HMGB1-mediated tumorigenesis. We found that in liver-specific autophagy-deficient (Atg7ΔHep) mice the tumors cells were still deficient in autophagy and could also release HMGB1. Histological analysis using cell-specific markers suggested that fibroblast and ductular cells were present only outside the tumor whereas macrophages were present both inside and outside the tumor. Genetic deletion of Hmgb1 or one of its receptors, receptor for advanced glycated end product (Rage), retarded liver tumor development. HMGB1 and RAGE enhanced the proliferation capability of the autophagy-deficient hepatocytes and tumors. However, RAGE expression was only found on ductual cells and Kupffer's cells but not on hepatoctyes, suggesting that HMGB1 might promote hepatic tumor growth through a paracrine mode, which altered the tumor microenvironment. Finally, RNAseq analysis of the tumors indicated that HMGB1 induced a much broad changes in tumors. In particular, genes related to mitochondrial structures or functions were enriched among those differentially expressed in tumors in the presence or absence of HMGB1, revealing a potentially important role of mitochondria in sustaining the growth of autophagy-deficient liver tumors via HMGB1 stimulation.

Role of High-Mobility Group Box-1 in Liver Pathogenesis.

High-mobility group box 1 (HMGB1) is a highly abundant DNA-binding protein that can relocate to the cytosol or undergo extracellular release during cellular stress or death. HMGB1 has a functional versatility depending on its cellular location. While intracellular HMGB1 is important for DNA structure maintenance, gene expression, and autophagy induction, extracellular HMGB1 acts as a damage-associated molecular pattern (DAMP) molecule to alert the host of damage by triggering immune responses. The biological function of HMGB1 is mediated by multiple receptors, including the receptor for advanced glycation end products (RAGE) and Toll-like receptors (TLRs), which are expressed in different hepatic cells. Activation of HMGB1 and downstream signaling pathways are contributing factors in the pathogenesis of non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), and drug-induced liver injury (DILI), each of which involves sterile inflammation, liver fibrosis, ductular reaction, and hepatic tumorigenesis. In this review, we will discuss the critical role of HMGB1 in these pathogenic contexts and propose HMGB1 as a bona fide and targetable DAMP in the setting of common liver diseases.

Autophagy, Metabolism, and Alcohol-Related Liver Disease: Novel Modulators and Functions.

Alcohol-related liver disease (ALD) is caused by over-consumption of alcohol. ALD can develop a spectrum of pathological changes in the liver, including steatosis, inflammation, cirrhosis, and complications. Autophagy is critical to maintain liver homeostasis, but dysfunction of autophagy has been observed in ALD. Generally, autophagy is considered to protect the liver from alcohol-induced injury and steatosis. In this review, we will summarize novel modulators of autophagy in hepatic metabolism and ALD, including autophagy-mediating non-coding RNAs (ncRNAs), and crosstalk of autophagy machinery and nuclear factors. We will also discuss novel functions of autophagy in hepatocytes and non-parenchymal hepatic cells during the pathogenesis of ALD and other liver diseases.

Hepatic senescence, the good and the bad.

Gradual alterations of cell's physiology and functions due to age or exposure to various stresses lead to the conversion of normal cells to senescent cells. Once becoming senescent, the cell stops dividing permanently but remains metabolically active. Cellular senescence does not have a single marker but is characterized mainly by a combination of multiple markers, such as, morphological changes, expression of cell cycle inhibitors, senescence associated ß-galactosidase activity, and changes in nuclear membrane. When cells in an organ become senescent, the entire organism can be affected. This may occur through the senescence-associated secretory phenotype (SASP). SASP may exert beneficial or harmful effects on the microenvironment of tissues. Research on senescence has become a very exciting field in cell biology since the link between age-related diseases, including cancer, and senescence has been established. The loss of regenerative and homeostatic capacity of the liver over the age is somehow connected to cellular senescence. The major contributors of senescence properties in the liver are hepatocytes and cholangiocytes. Senescent cells in the liver have been implicated in the etiology of chronic liver diseases including cirrhosis and hepatocellular carcinoma and in the interference of liver regeneration. This review summarizes recently reported findings in the understanding of the molecular mechanisms of senescence and its relationship with liver diseases.

Analysis of Autophagy for Liver Pathogenesis.

The autophagy pathway in hepatocytes is well characterized. Autophagy plays a critical role in the normal function of the liver. A growing number of studies suggest that there is a mechanistic relationship between autophagy and the pathogenesis of human diseases including liver diseases. Here we focus on the methods assessing the level of lipids, lipid peroxidation, and lipophagy in the liver, which would be particularly relevant to the study of fatty liver diseases.

Hepatic Autophagy Deficiency Compromises Farnesoid X Receptor Functionality and Causes Cholestatic Injury.

Autophagy is important for hepatic homeostasis, nutrient regeneration, and organelle quality control. We investigated the mechanisms by which liver injury occurred in the absence of autophagy function. We found that mice deficient in autophagy because of the lack of autophagy-related gene 7 or autophagy-related gene 5, key autophagy-related genes, manifested intracellular cholestasis with increased levels of serum bile acids, a higher ratio of tauromuricholic acid/taurocholic acid in the bile, increased hepatic bile acid load, abnormal bile canaliculi, and altered expression of hepatic transporters. In determining the underlying mechanism, we found that autophagy sustained and promoted the basal and up-regulated expression of farnesoid X receptor (Fxr) in the fed and starved conditions, respectively. Consequently, expression of Fxr and its downstream genes, particularly bile salt export pump, and the binding of FXR to the promoter regions of these genes, were suppressed in autophagy-deficient livers. In addition, codeletion of nuclear factor erythroid 2-related factor 2 (Nrf2) in autophagy deficiency status reversed the FXR suppression. Furthermore, the cholestatic injury of autophagy-deficient livers was reversed by enhancement of FXR activity or expression, or by Nrf2 deletion. Conclusion: Together with earlier reports that FXR can suppress autophagy, our findings indicate that autophagy and FXR form a regulatory loop and deficiency of autophagy causes abnormal FXR functionality, leading to the development of intracellular cholestasis and liver injury.

Autophagy is a gatekeeper of hepatic differentiation and carcinogenesis by controlling the degradation of Yap.

Activation of the Hippo pathway effector Yap underlies many liver cancers, however no germline or somatic mutations have been identified. Autophagy maintains essential metabolic functions of the liver, and autophagy-deficient murine models develop benign adenomas and hepatomegaly, which have been attributed to activation of the p62/Sqstm1-Nrf2 axis. Here, we show that Yap is an autophagy substrate and mediator of tissue remodeling and hepatocarcinogenesis independent of the p62/Sqstm1-Nrf2 axis. Hepatocyte-specific deletion of Atg7 promotes liver size, fibrosis, progenitor cell expansion, and hepatocarcinogenesis, which is rescued by concurrent deletion of Yap. Our results shed new light on mechanisms of Yap degradation and the sequence of events that follow disruption of autophagy, which is impaired in chronic liver disease.

Homeostatic Role of Autophagy in Hepatocytes.

Autophagy actively participates in the physiological process of the liver. While the direct effect of autophagy may be limited to the sequestration and degradation of a selective cargo, its overall impact can be broad, affecting many more physiological processes regulated by the particular cargo. This review will discuss two aspects of the importance of autophagy in the liver: metabolic regulation in response to feeding and starvation, and pathological consequences in the absence of autophagy. These two aspects illustrate the homeostatic functions of autophagy in the liver, one in a more direct fashion, regulating the cellular nutrient supply, and the other in a more indirect fashion, controlling the pathological signaling triggered by the abnormal accumulation of cargos. Remarkably, the hepatic pathology in autophagy-deficient livers does not seem different from that presented in other chronic liver diseases. Autophagy deficiency can be a model for the study of the relevant molecular mechanisms.

Dynamic MTORC1-TFEB feedback signaling regulates hepatic autophagy, steatosis and liver injury in long-term nutrient oversupply.

Normal metabolism requires a controlled balance between anabolism and catabolism. It is not completely known how this balance can be retained when the level of nutrient supply changes in the long term. We found that in murine liver anabolism, as represented by the phosphorylation of RPS6KB (ribosomal protein S6 kinase), was soon elevated while catabolism, as represented by TFEB (transcription factor EB)-directed gene transcription and lysosomal activities, was downregulated after the administration of a high-fat diet (HFD). Surprisingly, neither the alteration in RPS6KB phosphorylation nor that in TFEB functions was static over the long course of HFD feeding. Instead, the 2 signals exhibited dynamic alterations in opposite directions, which could be explained by the dependence of MTORC1 (MTOR complex 1) activation on TFEB-supported lysosome function and the feedback suppression of TFEB by MTORC1. Disruption of the dynamics by enforced expression of TFEB in HFD-fed mice at the peaks of MTORC1 activation restored lysosome function. Consistently, interference of MTORC1 activation with rapamycin or with a constitutively activated RRAGA mutant at the peak or nadir of MTORC1 oscillation enhanced or reduced the lysosome function, respectively. These treatments also improved or exacerbated hepatic steatosis and liver injury, respectively. Finally, there was a significant inverse correlation between TFEB activation and steatosis severity in the livers of patients with non-alcohol fatty liver diseases, supporting the clinical relevance of TFEB-regulated events. Thus, maintaining catabolic function through feedback mechanisms during enhanced anabolism, which is caused by nutrient oversupply, is important for reducing liver pathology.

HMGB1 promotes ductular reaction and tumorigenesis in autophagy-deficient livers.

Autophagy is important for liver homeostasis, and the deficiency leads to injury, inflammation, ductular reaction (DR), fibrosis, and tumorigenesis. It is not clear how these events are mechanistically linked to autophagy deficiency. Here, we reveal the role of high-mobility group box 1 (HMGB1) in two of these processes. First, HMGB1 was required for DR, which represents the expansion of hepatic progenitor cells (HPCs) implicated in liver repair and regeneration. DR caused by hepatotoxic diets (3,5-diethoxycarbonyl-1,4-dihydrocollidine [DDC] or choline-deficient, ethionine-supplemented [CDE]) also depended on HMGB1, indicating that HMGB1 may be generally required for DR in various injury scenarios. Second, HMGB1 promoted tumor progression in autophagy-deficient livers. Receptor for advanced glycation end product (RAGE), a receptor for HMGB1, was required in the same two processes and could mediate the proliferative effects of HMBG1 in isolated HPCs. HMGB1 was released from autophagy-deficient hepatocytes independently of cellular injury but depended on NRF2 and the inflammasome, which was activated by NRF2. Pharmacological or genetic activation of NRF2 alone, without disabling autophagy or causing injury, was sufficient to cause inflammasome-dependent HMGB1 release. In conclusion, HMGB1 release is a critical mechanism in hepatic pathogenesis under autophagy-deficient conditions and leads to HPC expansion as well as tumor progression.

Autophagy in non-alcoholic fatty liver disease and alcoholic liver disease.

Autophagy is an evolutionarily conserved intracellular degradative function that is important for liver homeostasis. Accumulating evidence suggests that autophagy is deregulated during the progression and development of alcoholic and non-alcoholic liver diseases. Impaired autophagy prevents the clearance of excessive lipid droplets (LDs), damaged mitochondria, and toxic protein aggregates, which can be generated during the progression of various liver diseases, thus contributing to the development of steatosis, injury, steatohepatitis, fibrosis, and tumors. In this review, we look at the status of hepatic autophagy during the pathogenesis of alcoholic and non-alcoholic liver diseases. We also examine the mechanisms of defects in autophagy, and the hepato-protective roles of autophagy in non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD), focusing mainly on steatosis and liver injury. Finally, we discuss the therapeutic potential of autophagy modulating agents for the treatment of these two common liver diseases.