Molecular cloning of the gene promoter encoding the human CaVγ2/Stargazin divergent transcript (CACNG2-DT): characterization and regulation by the cAMP-PKA/CREB signaling pathway.

CaVγ2 (Stargazin or TARPγ2) is a protein expressed in various types of neurons whose function was initially associated with a decrease in the functional expression of voltage-gated presynaptic Ca2+ channels (CaV) and which is now known to promote the trafficking of the postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPAR) towards the cell membrane. Alterations in CaVγ2 expression has been associated with several neurological disorders, such as absence epilepsy. However, its regulation at the transcriptional level has not been intensively addressed. It has been reported that the promoter of the Cacng2 gene, encoding the rat CaVγ2, is bidirectional and regulates the transcription of a long non-coding RNA (lncRNA) in the antisense direction. Here, we investigate the proximal promoter region of the human CACNG2 gene in the antisense direction and show that this region includes two functional cAMP response elements that regulate the expression of a lncRNA called CACNG2-DT. The activity of these sites is significantly enhanced by forskolin, an adenylate cyclase activator, and inhibited by H89, a protein kinase A (PKA) antagonist. Therefore, this regulatory mechanism implies the activation of G protein-coupled receptors and downstream phosphorylation. Interestingly, we also found that the expression of CACNG2-DT may increase the levels of the CaVγ2 subunit. Together, these data provide novel information on the organization of the human CACNG2-DT gene promoter, describe modulatory domains and mechanisms that can mediate various regulatory inputs, and provide initial information on the molecular mechanisms that regulate the functional expression of the CaVγ2 protein.

Cell-specific crosstalk proteomics reveals cathepsin B signaling as a driver of glioblastoma malignancy near the subventricular zone.

Glioblastoma (GBM) is the most prevalent and aggressive malignant primary brain tumor. GBM proximal to the lateral ventricles (LVs) is more aggressive, potentially due to subventricular zone (SVZ) contact. Despite this, crosstalk between GBM and neural stem/progenitor cells (NSC/NPCs) is not well understood. Using cell-specific proteomics, we show that LV-proximal GBM prevents neuronal maturation of NSCs through induction of senescence. Additionally, GBM brain tumor initiating cells (BTICs) increase expression of CTSB upon interaction with NPCs. Lentiviral knockdown and recombinant protein experiments reveal both cell-intrinsic and soluble CTSB promote malignancy-associated phenotypes in BTICs. Soluble CTSB stalls neuronal maturation in NPCs while promoting senescence, providing a link between LV-tumor proximity and neurogenesis disruption. Finally, we show LV-proximal CTSB upregulation in patients, showing the relevance of this crosstalk in human GBM biology. These results demonstrate the value of proteomic analysis in tumor microenvironment research and provide direction for new therapeutic strategies in GBM. Highlights: Periventricular GBM is more malignant and disrupts neurogenesis in a rodent model.Cell-specific proteomics elucidates tumor-promoting crosstalk between GBM and NPCs.NPCs induce upregulated CTSB expression in GBM, promoting tumor progression.GBM stalls neurogenesis and promotes NPC senescence via CTSB.

Development of Experimental Three-Dimensional Tumor Models to Study Glioblastoma Cancer Stem Cells and Tumor Microenvironment.

Glioblastoma (GBM) is the most common and dismal primary brain tumor. Unfortunately, despite multidisciplinary treatment, most patients will perish approximately 15 months after diagnosis. For this reason, there is an urgent need to improve our understanding of GBM tumor biology and develop novel therapies that can achieve better clinical outcomes. In this setting, three-dimensional tumor models have risen as more appropriate preclinical tools when compared to traditional cell cultures, given that two-dimensional (2D) cultures have failed to accurately recapitulate tumor biology and translate preclinical findings into patient benefits. Three-dimensional cultures using neurospheres, organoids, and organotypic better resemble original tumor genetic and epigenetic profiles, maintaining tumor microenvironment characteristics and mimicking cell-cell and cell-matrix interactions. This chapter summarizes our methods to generate well-characterized glioblastoma neurospheres, organoids, and organotypics.

Extracellular vesicles in the glioblastoma microenvironment: A diagnostic and therapeutic perspective.

Glioblastoma (GBM), is the most malignant form of gliomas and the most common and lethal primary brain tumor in adults. Conventional cancer treatments have limited to no efficacy on GBM. GBM cells respond and adapt to the surrounding brain parenchyma known as tumor microenvironment (TME) to promote tumor preservation. Among specific TME, there are 3 of particular interest for GBM biology: the perivascular niche, the subventricular zone neurogenic niche, and the immune microenvironment. GBM cells and TME cells present a reciprocal feedback which results in tumor maintenance. One way that these cells can communicate is through extracellular vesicles. These vesicles include exosomes and microvesicles that have the ability to carry both cancerous and non-cancerous cargo, such as miRNA, RNA, proteins, lipids, and DNA. In this review we will discuss the booming topic that is extracellular vesicles, and how they have the novelty to be a diagnostic and targetable vehicle for GBM.

Glioblastoma disrupts the ependymal wall and extracellular matrix structures of the subventricular zone.

BACKGROUND: Glioblastoma (GBM) is the most aggressive and common type of primary brain tumor in adults. Tumor location plays a role in patient prognosis, with tumors proximal to the lateral ventricles (LVs) presenting with worse overall survival, increased expression of stem cell genes, and increased incidence of distal tumor recurrence. This may be due in part to interaction of GBM with factors of the subventricular zone (SVZ), including those contained within the cerebrospinal fluid (CSF). However, direct interaction of GBM tumors with CSF has not been proved and would be hindered in the presence of an intact ependymal cell layer. METHODS: Here, we investigate the ependymal cell barrier and its derived extracellular matrix (ECM) fractones in the vicinity of a GBM tumor. Patient-derived GBM cells were orthotopically implanted into immunosuppressed athymic mice in locations distal and proximal to the LV. A PBS vehicle injection in the proximal location was included as a control. At four weeks post-xenograft, brain tissue was examined for alterations in ependymal cell health via immunohistochemistry, scanning electron microscopy, and transmission electron microscopy. RESULTS: We identified local invading GBM cells within the LV wall and increased influx of CSF into the LV-proximal GBM tumor bulk compared to controls. In addition to the physical disruption of the ependymal cell barrier, we also identified increased signs of compromised ependymal cell health in LV-proximal tumor-bearing mice. These signs include increased accumulation of lipid droplets, decreased cilia length and number, and decreased expression of cell channel proteins. We additionally identified elevated numbers of small fractones in the SVZ within this group, suggesting increased indirect CSF-contained molecule signaling to tumor cells. CONCLUSIONS: Our data is the first to show that LV-proximal GBMs physically disrupt the ependymal cell barrier in animal models, resulting in disruptions in ependymal cell biology and increased CSF interaction with the tumor bulk. These findings point to ependymal cell health and CSF-contained molecules as potential axes for therapeutic targeting in the treatment of GBM.

Activation of STAT3 through combined SRC and EGFR signaling drives resistance to a mitotic kinesin inhibitor in glioblastoma.

Inhibitors of the mitotic kinesin Kif11 are anti-mitotics that, unlike vinca alkaloids or taxanes, do not disrupt microtubules and are not neurotoxic. However, development of resistance has limited their clinical utility. While resistance to Kif11 inhibitors in other cell types is due to mechanisms that prevent these drugs from disrupting mitosis, we find that in glioblastoma (GBM), resistance to the Kif11 inhibitor ispinesib works instead through suppression of apoptosis driven by activation of STAT3. This form of resistance requires dual phosphorylation of STAT3 residues Y705 and S727, mediated by SRC and epidermal growth factor receptor (EGFR), respectively. Simultaneously inhibiting SRC and EGFR reverses this resistance, and combined targeting of these two kinases in vivo with clinically available inhibitors is synergistic and significantly prolongs survival in ispinesib-treated GBM-bearing mice. We thus identify a translationally actionable approach to overcoming Kif11 inhibitor resistance that may work to block STAT3-driven resistance against other anti-cancer therapies as well.

Functional Characterization of Brain Tumor-Initiating Cells and Establishment of GBM Preclinical Models that Incorporate Heterogeneity, Therapy, and Sex Differences.

Glioblastoma (GBM) is the most common primary brain cancer in adults where tumor cell heterogeneity and sex differences influence clinical outcomes. Here, we functionally characterize three male and three female patient-derived GBM cell lines, identify protumorigenic BTICs, and create novel male and female preclinical models of GBM. Cell lines were evaluated on the following features: proliferation, stemness, migration, tumorigenesis, clinical characteristics, and sensitivity to radiation, TMZ, rhTNFSF10 (rhTRAIL), and rhBMP4 All cell lines were classified as GBM according to epigenetic subtyping, were heterogenous and functionally distinct from one another, and re-capitulated features of the original patient tumor. In establishing male and female preclinical models, it was found that two male-derived GBM cell lines (QNS108 and QNS120) and one female-derived GBM cell line (QNS315) grew at a faster rate in female mice brains. One male-derived GBM cell line (QNS108) decreased survival in female mice in comparison with male mice. However, no survival differences were observed for mice injected with a female-derived cell line (QNS315). In summary, a panel of six GBM patient-derived cell lines were functionally characterized, and it was shown that BTIC lines can be used to construct sex-specific models with differential phenotypes for additional studies.

Glioblastoma Proximity to the Lateral Ventricle Alters Neurogenic Cell Populations of the Subventricular Zone.

Despite current strategies combining surgery, radiation, and chemotherapy, glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor in adults. Tumor location plays a key role in the prognosis of patients, with GBM tumors located in close proximity to the lateral ventricles (LVs) resulting in worse survival expectancy and higher incidence of distal recurrence. Though the reason for worse prognosis in these patients remains unknown, it may be due to proximity to the subventricular zone (SVZ) neurogenic niche contained within the lateral wall of the LVs. We present a novel rodent model to analyze the bidirectional signaling between GBM tumors and cells contained within the SVZ. Patient-derived GBM cells expressing GFP and luciferase were engrafted at locations proximal, intermediate, and distal to the LVs in immunosuppressed mice. Mice were either sacrificed after 4 weeks for immunohistochemical analysis of the tumor and SVZ or maintained for survival analysis. Analysis of the GFP+ tumor bulk revealed that GBM tumors proximal to the LV show increased levels of proliferation and tumor growth than LV-distal counterparts and is accompanied by decreased median survival. Conversely, numbers of innate proliferative cells, neural stem cells (NSCs), migratory cells and progenitors contained within the SVZ are decreased as a result of GBM proximity to the LV. These results indicate that our rodent model is able to accurately recapitulate several of the clinical aspects of LV-associated GBM, including increased tumor growth and decreased median survival. Additionally, we have found the neurogenic and cell division process of the SVZ in these adult mice is negatively influenced according to the presence and proximity of the tumor mass. This model will be invaluable for further investigation into the bidirectional signaling between GBM and the neurogenic cell populations of the SVZ.

Phosphorylated WNK kinase networks in recoded bacteria recapitulate physiological function.

Advances in genetic code expansion have enabled the production of proteins containing site-specific, authentic post-translational modifications. Here, we use a recoded bacterial strain with an expanded genetic code to encode phosphoserine into a human kinase protein. We directly encode phosphoserine into WNK1 (with-no-lysine [K] 1) or WNK4 kinases at multiple, distinct sites, which produced activated, phosphorylated WNK that phosphorylated and activated SPAK/OSR kinases, thereby synthetically activating this human kinase network in recoded bacteria. We used this approach to identify biochemical properties of WNK kinases, a motif for SPAK substrates, and small-molecule kinase inhibitors for phosphorylated SPAK. We show that the kinase inhibitors modulate SPAK substrates in cells, alter cell volume, and reduce migration of glioblastoma cells. Our work establishes a protein-engineering platform technology that demonstrates that synthetically active WNK kinase networks can accurately model cellular systems and can be used more broadly to target networks of phosphorylated proteins for research and discovery.

Human Cerebrospinal Fluid Modulates Pathways Promoting Glioblastoma Malignancy.

Glioblastoma (GBM) is the most common and devastating primary cancer of the central nervous system in adults. High grade gliomas are able to modify and respond to the brain microenvironment. When GBM tumors infiltrate the Subventricular zone (SVZ) they have a more aggressive clinical presentation than SVZ-distal tumors. We suggest that cerebrospinal fluid (CSF) contact contributes to enhance GBM malignant characteristics in these tumors. We evaluated the impact of human CSF on GBM, performing a transcriptome analysis on human primary GBM cells exposed to CSF to measure changes in gene expression profile and their clinical relevance on disease outcome. In addition we evaluated the proliferation and migration changes of CSF-exposed GBM cells in vitro and in vivo. CSF induced transcriptomic changes in pathways promoting cell malignancy, such as apoptosis, survival, cell motility, angiogenesis, inflammation, and glucose metabolism. A genetic signature extracted from the identified transcriptional changes in response to CSF proved to be predictive of GBM patient survival using the TCGA database. Furthermore, CSF induced an increase in viability, proliferation rate, and self-renewing capacity, as well as the migratory capabilities of GBM cells in vitro. In vivo, GBM cells co-injected with human CSF generated larger and more proliferative tumors compared to controls. Taken together, these results provide direct evidence that CSF is a key player in determining tumor growth and invasion through the activation of complex gene expression patterns characteristic of a malignant phenotype. These findings have diagnostic and therapeutic implications for GBM patients. The changes induced by CSF contact might play a role in the increased malignancy of SVZ-proximal GBM.

Circulatory shear stress induces molecular changes and side population enrichment in primary tumor-derived lung cancer cells with higher metastatic potential.

Cancer is a leading cause of death and disease worldwide. However, while the survival for patients with primary cancers is improving, the ability to prevent metastatic cancer has not. Once patients develop metastases, their prognosis is dismal. A critical step in metastasis is the transit of cancer cells in the circulatory system. In this hostile microenvironment, variations in pressure and flow can change cellular behavior. However, the effects that circulation has on cancer cells and the metastatic process remain unclear. To further understand this process, we engineered a closed-loop fluidic system to analyze molecular changes induced by variations in flow rate and pressure on primary tumor-derived lung adenocarcinoma cells. We found that cancer cells overexpress epithelial-to-mesenchymal transition markers TWIST1 and SNAI2, as well as stem-like marker CD44 (but not CD133, SOX2 and/or NANOG). Moreover, these cells display a fourfold increased percentage of side population cells and have an increased propensity for migration. In vivo, surviving circulatory cells lead to decreased survival in rodents. These results suggest that cancer cells that express a specific circulatory transition phenotype and are enriched in side population cells are able to survive prolonged circulatory stress and lead to increased metastatic disease and shorter survival.

Alpha 1-antichymotrypsin contributes to stem cell characteristics and enhances tumorigenicity of glioblastoma.

BACKGROUND: Glioblastomas (GBMs) are the main primary brain tumors in adults with almost 100% recurrence rate. Patients with lateral ventricle proximal GBMs (LV-GBMs) exhibit worse survival compared to distal locations for unknown reasons. One hypothesis is the proximity of these tumors to the cerebrospinal fluid (CSF) and its chemical cues that can regulate cellular phenotype. We therefore investigated the role of CSF on GBM gene expression and the role of a CSF-induced gene, SERPINA3, in GBM malignancy in vitro and in vivo. METHODS: We utilized human CSF and GBM brain tumor-initiating cells (BTICs). We determined the impact of SERPINA3 expression in glioma patients using The Cancer Genome Atlas (TCGA) database. SERPINA3 expression changes were evaluated at mRNA and protein levels. The effects of knockdown (KD) and overexpression (OE) of SERPINA3 on cell migration, viability and cell proliferation were evaluated. Stem cell characteristics on KD cells were evaluated by differentiation and colony formation experiments. Tumor growth was studied by intracranial and flank injections. RESULTS: GBM-CSF increased BTIC migration accompanied by upregulation of the SERPINA3 gene. In patient samples and TCGA data, we observed SERPINA3 to correlate directly with brain tumor grade and indirectly with GBM patient survival. SERPINA3 KD induced a decrease in cell proliferation, migration, invasion, and stem cell characteristics, while SERPINA3 OE increased cell migration. In vivo, SERPINA3 KD BTICs showed increased survival in a murine model. CONCLUSIONS: SERPINA3 plays a key role in GBM malignancy and its inhibition results in a better outcome using GBM preclinical models.

Myosin 10 Regulates Invasion, Mitosis, and Metabolic Signaling in Glioblastoma.

Invasion and proliferation are defining phenotypes of cancer, and in glioblastoma blocking one stimulates the other, implying that effective therapy must inhibit both, ideally through a single target that is also dispensable for normal tissue function. The molecular motor myosin 10 meets these criteria. Myosin 10 knockout mice can survive to adulthood, implying that normal cells can compensate for its loss; its deletion impairs invasion, slows proliferation, and prolongs survival in murine models of glioblastoma. Myosin 10 deletion also enhances tumor dependency on the DNA damage and the metabolic stress responses and induces synthetic lethality when combined with inhibitors of these processes. Our results thus demonstrate that targeting myosin 10 is active against glioblastoma by itself, synergizes with other clinically available therapeutics, may have acceptable side effects in normal tissues, and has potential as a heretofore unexplored therapeutic approach for this disease.

Regulation of the Ca2+ channel α2δ-1 subunit expression by epidermal growth factor via the ERK/ELK-1 signaling pathway.

Voltage-gated Ca2+ (CaV) channels are expressed in endocrine cells where they contribute to hormone secretion. Diverse chemical messengers, including epidermal growth factor (EGF), are known to affect the expression of CaV channels. Previous studies have shown that EGF increases Ca2+ currents in GH3 pituitary cells by increasing the number of high voltage-activated (HVA) CaV channels at the cell membrane, which results in enhanced prolactin (PRL) secretion. However, little is known regarding the mechanisms underlying this regulation. Here, we show that EGF actually increases the expression of the CaVα2δ-1 subunit, a key molecular component of HVA channels. The analysis of the gene promoter encoding CaVα2δ-1 (CACNA2D1) revealed binding sites for transcription factors activated by the Ras/Raf/MEK/ERK signaling cascade. Chromatin immunoprecipitation and site-directed mutagenesis showed that ELK-1 is crucial for the transcriptional regulation of CACNA2D1 in response to EGF. Furthermore, we found that EGF increases the membrane expression of CaVα2δ-1 and that ELK-1 overexpression increases HVA current density, whereas ELK-1 knockdown decreases the functional expression of the channels. Hormone release assays revealed that CaVα2δ-1 overexpression increases PRL secretion. These results suggest a mechanism for how EGF, by activating the Ras/Raf/MEK/ERK/ELK-1 pathway, may influence the expression of HVA channels and the secretory behavior of pituitary cells.

Phospho-Tau Protein Expression in the Cell Cycle of SH-SY5Y Neuroblastoma Cells: A Morphological Study.

It has been reported that the main function of tau protein is to stabilize microtubules and promote the movement of organelles through the axon in neurons. In Alzheimer's disease, tau protein is the major constituent of the paired helical filament, and it undergoes post-translational modifications including hyperphosphorylation and truncation. Whether other functions of tau protein are involved in Alzheimer's disease is less clear. We used SH-SY5Y human neuroblastoma cells as an in vitro model to further study the functions of tau protein. We detected phosphorylated tau protein as small dense dots in the cell nucleus, which strongly colocalize with intranuclear speckle structures that were also labelled with an antibody to SC35, a protein involved in nuclear RNA splicing. We have shown further that tau protein, phosphorylated at the sites recognized by pT231, TG-3, and AD2 antibodies, is closely associated with cell division. Different functions may be characteristic of phosphorylation at specific sites. Our findings suggest that the presence of tau protein is involved in separation of sister chromatids in anaphase, and that tau protein also participates in maintaining the integrity of the DNA (pT231, prophase) and chromosomes during cell division (TG-3).

Overlapping migratory mechanisms between neural progenitor cells and brain tumor stem cells.

Neural stem cells present in the subventricular zone (SVZ), the largest neurogenic niche of the mammalian brain, are able to self-renew as well as generate neural progenitor cells (NPCs). NPCs are highly migratory and traverse the rostral migratory stream (RMS) to the olfactory bulb, where they terminally differentiate into mature interneurons. NPCs from the SVZ are some of the few cells in the CNS that migrate long distances during adulthood. The migratory process of NPCs is highly regulated by intracellular pathway activation and signaling from the surrounding microenvironment. It involves modulation of cell volume, cytoskeletal rearrangement, and isolation from compact extracellular matrix. In malignant brain tumors including high-grade gliomas, there are cells called brain tumor stem cells (BTSCs) with similar stem cell characteristics to NPCs but with uncontrolled cell proliferation and contribute to tumor initiation capacity, tumor progression, invasion, and tumor maintenance. These BTSCs are resistant to chemotherapy and radiotherapy, and their presence is believed to lead to tumor recurrence at distal sites from the original tumor location, principally due to their high migratory capacity. BTSCs are able to invade the brain parenchyma by utilizing many of the migratory mechanisms used by NPCs. However, they have an increased ability to infiltrate the tight brain parenchyma and utilize brain structures such as myelin tracts and blood vessels as migratory paths. In this article, we summarize recent findings on the mechanisms of cellular migration that overlap between NPCs and BTSCs. A better understanding of the intersection between NPCs and BTSCs will to provide a better comprehension of the BTSCs' invasive capacity and the molecular mechanisms that govern their migration and eventually lead to the development of new therapies to improve the prognosis of patients with malignant gliomas.

Expression of Gas1 in Mouse Brain: Release and Role in Neuronal Differentiation.

Growth arrest-specific 1 (Gas1) is a pleiotropic protein that induces apoptosis of tumor cells and has important roles during development. Recently, the presence of two forms of Gas1 was reported: one attached to the cell membrane by a GPI anchor; and a soluble extracellular form shed by cells. Previously, we showed that Gas1 is expressed in different areas of the adult mouse CNS. Here, we report the levels of Gas1 mRNA protein in different regions and analyzed its expressions in glutamatergic, GABAergic, and dopaminergic neurons. We found that Gas1 is expressed in GABAergic and glutamatergic neurons in the Purkinje-molecular layer of the cerebellum, hippocampus, thalamus, and fastigial nucleus, as well as in dopaminergic neurons of the substantia nigra. In all cases, Gas1 was found in the cell bodies, but not in the neuropil. The Purkinje and the molecular layers show the highest levels of Gas1, whereas the granule cell layer has low levels. Moreover, we detected the expression and release of Gas1 from primary cultures of Purkinje cells and from hippocampal neurons as well as from neuronal cell lines, but not from cerebellar granular cells. In addition, using SH-SY5Y cells differentiated with retinoic acid as a neuronal model, we found that extracellular Gas1 promotes neurite outgrowth, increases the levels of tyrosine hydroxylase, and stimulates the inhibition of GSK3ß. These findings demonstrate that Gas1 is expressed and released by neurons and promotes differentiation, suggesting an important role for Gas1 in cellular signaling in the CNS.

Naringenin prevents experimental liver fibrosis by blocking TGFß-Smad3 and JNK-Smad3 pathways.

AIM: To study the molecular mechanisms involved in the hepatoprotective effects of naringenin (NAR) on carbon tetrachloride (CCl4)-induced liver fibrosis. METHODS: Thirty-two male Wistar rats (120-150 g) were randomly divided into four groups: (1) a control group (n = 8) that received 0.7% carboxy methyl-cellulose (NAR vehicle) 1 mL/daily p.o.; (2) a CCl4 group (n = 8) that received 400 mg of CCl4/kg body weight i.p. 3 times a week for 8 wk; (3) a CCl4 + NAR (n = 8) group that received 400 mg of CCl4/kg body weight i.p. 3 times a week for 8 wk and 100 mg of NAR/kg body weight daily for 8 wk p.o.; and (4) an NAR group (n = 8) that received 100 mg of NAR/kg body weight daily for 8 wk p.o. After the experimental period, animals were sacrificed under ketamine and xylazine anesthesia. Liver damage markers such as alanine aminotransferase (ALT), alkaline phosphatase (AP), γ-glutamyl transpeptidase (γ-GTP), reduced glutathione (GSH), glycogen content, lipid peroxidation (LPO) and collagen content were measured. The enzymatic activity of glutathione peroxidase (GPx) was assessed. Liver histopathology was performed utilizing Masson's trichrome and hematoxylin-eosin stains. Zymography assays for MMP-9 and MMP-2 were carried out. Hepatic TGF-ß, α-SMA, CTGF, Col-I, MMP-13, NF-κB, IL-1, IL-10, Smad7, Smad3, pSmad3 and pJNK proteins were detected via western blot. RESULTS: NAR administration prevented increases in ALT, AP, γ-GTP, and GPx enzymatic activity; depletion of GSH and glycogen; and increases in LPO and collagen produced by chronic CCl4 intoxication (P < 0.05). Liver histopathology showed a decrease in collagen deposition when rats received NAR in addition to CCl4. Although zymography assays showed that CCl4 produced an increase in MMP-9 and MMP-2 gelatinase activity; interestingly, NAR administration was associated with normal MMP-9 and MMP-2 activity (P < 0.05). The anti-inflammatory, antinecrotic and antifibrotic effects of NAR may be attributed to its ability to prevent NF-κB activation and the subsequent production of IL-1 and IL-10 (P < 0.05). NAR completely prevented the increase in TGF-ß, α-SMA, CTGF, Col-1, and MMP-13 proteins compared with the CCl4-treated group (P < 0.05). NAR prevented Smad3 phosphorylation in the linker region by JNK since this flavonoid blocked this kinase (P < 0.05). CONCLUSION: NAR prevents CCl4 induced liver inflammation, necrosis and fibrosis, due to its antioxidant capacity as a free radical inhibitor and by inhibiting the NF-κB, TGF-ß-Smad3 and JNK-Smad3 pathways.

Coffee consumption prevents fibrosis in a rat model that mimics secondary biliary cirrhosis in humans.

Investigations demonstrated that oxidative stress plays an important role in injury promotion in cholestatic liver disease. We hypothesized that coffee attenuates cholestasis-induced hepatic necrosis and fibrosis via its antioxidant, anti-inflammatory, and antifibrotic properties. The major aim of this study was to evaluate the hepatoprotective properties of coffee and caffeine in a model of chronic bile duct ligation (BDL) in male Wistar rats. Liver injury was induced by 28-day BDL, and conventional coffee, decaffeinated coffee, or caffeine was administered daily. After treatment, the hepatic oxidative status was estimated by measuring lipid peroxidation, the reduced to oxidized glutathione ratio, and glutathione peroxidase. Fibrosis was assessed by measuring the liver hydroxyproline content. The transforming growth factor-ß, connective tissue growth factor, α-smooth muscle actin, collagen 1, and interleukin-10 proteins and mRNAs were measured by Western blot and polymerase chain reaction, respectively. Conventional coffee suppressed most of the changes produced by BDL; however, caffeine showed better antifibrotic effects. Coffee demonstrated antioxidant properties by restoring the redox equilibrium, and it also prevented the elevation of liver enzymes as well as hepatic glycogen depletion. Interestingly, coffee and caffeine administration prevented collagen increases. Western blot assays showed decreased expression levels of transforming growth factor-ß, connective tissue growth factor, α-smooth muscle actin, and collagen 1 in the coffee- and caffeine-treated BDL groups. Similarly, coffee decreased the mRNA levels of these proteins. We conclude that coffee prevents liver cirrhosis induced by BDL by attenuating the oxidant processes, blocking hepatic stellate cell activation, and downregulating the main profibrotic molecules involved in extracellular matrix deposition.