Per- and polyfluoroalkyl substances activate UPR pathway, induce steatosis and fibrosis in liver cells.

Per- and polyfluoroalkyl substances (PFAS), which include perfluorooctanoic acid (PFOA), heptafluorobutyric acid (HFBA), and perfluorotetradecanoic acid (PFTA), are commonly occurring organic pollutants. Exposure to PFAS affects the immune system, thyroid and kidney function, lipid metabolism, and insulin signaling and is also involved in the development of fatty liver disease and cancer. The molecular mechanisms by which PFAS cause fatty liver disease are not understood in detail. In the current study, we investigated the effect of low physiologically relevant concentrations of PFOA, HFBA, and PFTA on cell survival, steatosis, and fibrogenic signaling in liver cell models. Exposure of PFOA and HFBA (10 to 1000 nM) specifically promoted cell survival in HepaRG and HepG2 cells. PFAS increased the expression of TNFα and IL6 inflammatory markers, increased endogenous reactive oxygen species (ROS) production, and activated unfolded protein response (UPR). Furthermore, PFAS enhanced cell steatosis and fibrosis in HepaRG and HepG2 cells which were accompanied by upregulation of steatosis (SCD1, ACC, SRBP1, and FASN), and fibrosis (TIMP2, p21, TGFß) biomarkers expression, respectively. RNA-seq data suggested that chronic exposures to PFOA modulated the expression of fatty acid/lipid metabolic genes that are involved in the development of NFALD and fatty liver disease. Collectively our data suggest that acute/chronic physiologically relevant concentrations of PFAS enhance liver cell steatosis and fibrosis by the activation of the UPR pathway and by modulation of NFALD-related gene expression.

Cadmium modulates steatosis, fibrosis, and oncogenic signaling in liver cancer cells by activating notch and AKT/mTOR pathways.

Cadmium (Cd) is an environmental pollutant that increases hepatotoxicity and the risk of liver diseases. In the current study, we investigated the effect of a physiologically relevant, low concentration of Cd on the regulation of liver cancer cell proliferation, steatosis, and fibrogenic/oncogenic signaling. Exposure to low concentrations of Cd increased endogenous reactive oxygen species (ROS) production and enhanced cell proliferation in a human bipotent progenitor cell line HepaRG and hepatocellular carcinoma (HCC) cell lines. Acute exposure of Cd increased Jagged-1 expression and activated Notch signaling in HepaRG and HCC cells HepG2 and SK-Hep1. Cd activated AKT/mTOR signaling by increasing phosphorylation of AKT-S473 and mTOR-S-4448 residues. Moreover, a low concentration of Cd also promoted cell steatosis and induced fibrogenic signaling in HCC cells. Chronic exposure to low concentrations of Cd-activated Notch and AKT/mTOR signaling induced the expression of pro-inflammatory cytokines tumor necrosis factor-alpha (TNFα) and its downstream target TNF-α-Induced Protein 8 (TNFAIP8). RNA-Seq data revealed that chronic exposure to low concentrations of Cd modulated the expression of several fatty liver disease-related genes involved in cell steatosis/fibrosis in HepaRG and HepG2 cells. Collectively, our data suggest that low concentrations of Cd modulate steatosis along with fibrogenic and oncogenic signaling in HCC cells by activating Notch and AKT/mTOR pathways.

MicroRNA-99b-5p targets mTOR/AR axis, induces autophagy and inhibits prostate cancer cell proliferation.

OBJECTIVES: MicroRNAs (miRNAs) are the small non-coding regulatory RNA molecules involved in gene regulation via base-pairing with complementary sequences in mRNAs. The dysregulation of specific miRNAs, such as miR-99b-5p (miR-99b), is associated with prostate cancer (PCa) progression. However, the mechanistic role of miR-99b in PCa remains to be determined. In this study, we aimed to investigate the functional and clinical significance of miR-99b in PCa. STUDY DESIGN: The expression of miR-99b and its downstream targets mTOR/AR in the PCa samples were analyzed by RT/qPCR. The effects of miR-99b overexpression/inhibition on PCa cell survival/proliferation, spheroid formation, and cell migration were examined by specific assays. Luciferase reporter assays were performed to determine the binding of miR-99b to 3' untranslated region (UTR) of the mTOR gene. The effects of miR-99b on the expression of mTOR, AR, and PSA proteins, as well as on AKT/mTOR signaling, autophagy, and neuroendocrine differentiation markers were analyzed by western blotting. The expression of miR-99b, mTOR, AR, PSA in AR-negative PC3 and AR-positive LNCaP cells was analyzed by RT/qPCR. The effect of miR-99b on global gene expression in PC3 cells was analyzed by RNA-seq. RESULTS: The expression of miR-99b was downregulated in tumor samples from PCa patients, whereas the expression of mTOR and AR was upregulated. In PCa cell lines, overexpression of miR-99b inhibited cell proliferation and cell colony/spheroid formation; induced apoptosis, and increased sensitivity towards docetaxel (DTX). In contrast, inhibition of miR-99b by miR-99b inhibitor resulted in increased cell growth in PCa cells. Mechanistically, miR-99b inhibited the expression of the mammalian target of the rapamycin (mTOR) gene by binding to its 3' UTR and induced autophagy. Furthermore, miR-99b inhibited androgen receptor (AR) activity in LNCaP cells and induced apoptosis. Activation of AR signaling by dihydrotestosterone (DHT) downregulated miR-99b expression and promoted cell PCa cell growth/survival, whereas inactivation of mTOR by rapamycin or AR by enzalutamide decreased miR-99b mediated PCa cell growth. CONCLUSION: Our data suggest that miR-99b functions as a tumor suppressor by targeting the mTOR/AR axis in PCa cells, implicating miR-99b as a novel biomarker and therapeutic target for PCa management.

Viral persistence, liver disease, and host response in a hepatitis C-like virus rat model.

The lack of a relevant, tractable, and immunocompetent animal model for hepatitis C virus (HCV) has severely impeded investigations of viral persistence, immunity, and pathogenesis. In the absence of immunocompetent models with robust HCV infection, homolog hepaciviruses in their natural host could potentially provide useful surrogate models. We isolated a rodent hepacivirus from wild rats (Rattus norvegicus), RHV-rn1; acquired the complete viral genome sequence; and developed an infectious reverse genetics system. RHV-rn1 resembles HCV in genomic features including the pattern of polyprotein cleavage sites and secondary structures in the viral 5' and 3' untranslated regions. We used site-directed and random mutagenesis to determine that only the first of the two microRNA-122 seed sites in the viral 5' untranslated region is required for viral replication and persistence in rats. Next, we used the clone-derived virus progeny to infect several inbred and outbred rat strains. Our results determined that RHV-rn1 possesses several HCV-defining hallmarks: hepatotropism, propensity to persist, and the ability to induce gradual liver damage. Histological examination of liver samples revealed the presence of lymphoid aggregates, parenchymal inflammation, and macrovesicular and microvesicular steatosis in chronically infected rats. Gene expression analysis demonstrated that the intrahepatic response during RHV-rn1 infection in rats mirrors that of HCV infection, including persistent activation of interferon signaling pathways. Finally, we determined that the backbone drug of HCV direct-acting antiviral therapy, sofosbuvir, effectively suppresses chronic RHV-rn1 infection in rats. CONCLUSION: We developed RHV-rn1-infected rats as a fully immunocompetent and informative surrogate model to delineate the mechanisms of HCV-related viral persistence, immunity, and pathogenesis. (Hepatology 2018).

Virus discovery reveals frequent infection by diverse novel members of the Flaviviridae in wild lemurs.

Lemurs are highly endangered mammals inhabiting the forests of Madagascar. In this study, we performed virus discovery on serum samples collected from 84 wild lemurs and identified viral sequence fragments from 4 novel viruses within the family Flaviviridae, including members of the genera Hepacivirus and Pegivirus. The sifaka hepacivirus (SifHV, two genotypes) and pegivirus (SifPgV, two genotypes) were discovered in the diademed sifaka (Propithecus diadema), while other pegiviral fragments were detected in samples from the indri (Indri indri, IndPgV) and the weasel sportive lemur (Lepilemur mustelinus, LepPgV). Although data are preliminary, each viral species appeared host species-specific and frequent infection was detected (18 of 84 individuals were positive for at least one virus). The complete coding sequence and partial 5' and 3' untranslated regions (UTRs) were obtained for SifHV and its genomic organization was consistent with that of other hepaciviruses, with one unique polyprotein and highly structured UTRs. Phylogenetic analyses showed the SifHV belonged to a clade that includes several viral species identified in rodents from Asia and North America, while SifPgV and IndPgV were more closely related to pegiviral species A and C, that include viruses found in humans as well as New- and Old-World monkeys. Our results support the current proposed model of virus-host co-divergence with frequent occurrence of cross-species transmission for these genera and highlight how the discovery of more members of the Flaviviridae can help clarify the ecology and evolutionary history of these viruses. Furthermore, this knowledge is important for conservation and captive management of lemurs.

Clemastine and hyperthermia enhance sensitization of osteosarcoma cells for apoptosis.

Clemastine is an antagonist of histamine H1 receptor may provide benefits in the treatment of osteosarcoma (OS). In the current study, we used hyperthermia approach to sensitize OS cells to clemastine-mediated cell death. Osteosarcoma U-2 OS and Saos-2 cells were treated with clemastine at 37°C, followed by 42°C for 2 h, and released at 37°C for 6 h. The impact of clemastine and hyperthermia on OS cell survival and autophagy-mediated cell death was investigated. Exposure of U-2 OS and Saos-2 cells to clemastine and hyperthermia (42°C) inhibited dose-dependent clemastine-mediated cell survival by increasing cell apoptosis. Hyperthermia and clemastine exposure modulated inflammatory and unfolded protein response (UPR) signaling differentially in U-2 OS and Saos-2 cells. Exposure of U-2 OS and Saos-2 cells to hyperthermia and clemastine inhibited AKT/mTOR and induced expression of the autophagy biomarkers LC3B II and LC3-positive puncta formation. The inhibition of autophagy by 3-methyladenine blocked hyperthermia and clemastine-mediated induction of LC3B II, LC3-positive puncta formation, and OS cell apoptosis. These results indicate that clemastine and hyperthermia sensitize OS cell lines by inducing increased autophagic cell death. Collectively, our data suggest that hyperthermia along with antihistamine therapy may provide an improved approach for the treatment of OS.

Cyanotoxins Increase Cytotoxicity and Promote Nonalcoholic Fatty Liver Disease Progression by Enhancing Cell Steatosis.

Freshwater prokaryotic cyanobacteria within harmful algal blooms produce cyanotoxins which are considered major pollutants in the aquatic system. Direct exposure to cyanotoxins through inhalation, skin contact, or ingestion of contaminated drinking water can target the liver and may cause hepatotoxicity. In the current study, we investigated the effect of low concentrations of cyanotoxins on cytotoxicity, inflammation, modulation of unfolded protein response (UPR), steatosis, and fibrosis signaling in human hepatocytes and liver cell models. Exposure to low concentrations of microcystin-LR (MC-LR), microcystin-RR (MC-RR), nodularin (NOD), and cylindrospermopsin (CYN) in human bipotent progenitor cell line HepaRG and hepatocellular carcinoma (HCC) cell lines HepG2 and SK-Hep1 resulted in increased cell toxicity. MC-LR, NOD, and CYN differentially regulated inflammatory signaling, activated UPR signaling and lipogenic gene expression, and induced cellular steatosis and fibrotic signaling in HCC cells. MC-LR, NOD, and CYN also regulated AKT/mTOR signaling and inhibited autophagy. Chronic exposure to MC-LR, NOD, and CYN upregulated the expression of lipogenic and fibrosis biomarkers. Moreover, RNA sequencing (RNA seq) data suggested that exposure of human hepatocytes, HepaRG, and HCC HepG2 cells to MC-LR and CYN modulated expression levels of several genes that regulate non-alcoholic fatty liver disease (NAFLD). Our data suggest that low concentrations of cyanotoxins can cause hepatotoxicity and cell steatosis and promote NAFLD progression.

MicroRNA-483-5p Inhibits Hepatocellular Carcinoma Cell Proliferation, Cell Steatosis, and Fibrosis by Targeting PPARα and TIMP2.

MicroRNAs (miRNAs) are small non-coding RNA molecules that bind with the 3' untranslated regions (UTRs) of genes to regulate expression. Downregulation of miR-483-5p (miR-483) is associated with the progression of hepatocellular carcinoma (HCC). However, the significant roles of miR-483 in nonalcoholic fatty liver disease (NAFLD), alcoholic fatty liver diseases (AFLD), and HCC remain elusive. In the current study, we investigated the biological significance of miR-483 in NAFLD, AFLD, and HCC in vitro and in vivo. The downregulation of miR-483 expression in HCC patients' tumor samples was associated with Notch 3 upregulation. Overexpression of miR-483 in a human bipotent progenitor liver cell line HepaRG and HCC cells dysregulated Notch signaling, inhibited cell proliferation/migration, induced apoptosis, and increased sensitivity towards antineoplastic agents sorafenib/regorafenib. Interestingly, the inactivation of miR-483 upregulated cell steatosis and fibrosis signaling by modulation of lipogenic and fibrosis gene expression. Mechanistically, miR-483 targets PPARα and TIMP2 gene expression, which leads to the suppression of cell steatosis and fibrosis. The downregulation of miR-483 was observed in mice liver fed with a high-fat diet (HFD) or a standard Lieber-Decarli liquid diet containing 5% alcohol, leading to increased hepatic steatosis/fibrosis. Our data suggest that miR-483 inhibits cell steatosis and fibrogenic signaling and functions as a tumor suppressor in HCC. Therefore, miR-483 may be a novel therapeutic target for NAFLD/AFLD/HCC management in patients with fatty liver diseases and HCC.

Deficiency of spns1 exacerbates per- and polyfluoroalkyl substances mediated hepatic toxicity and steatosis in zebrafish (Danio rerio).

Per- and polyfluoroalkyl substances (PFAS) are man-made long-lasting chemical compounds that are found in everyday household items. Today they occur in the environment as a major group of pollutants. These compounds are broadly used in commercial product preparation such as, for food packaging, nonstick coatings, and firefighting foam. In humans, PFAS can cause immune disorders, impaired fetal development, abnormal skeletal tissue development, osteoarthritis, thyroid dysfunctions, cholesterol changes, affect insulin regulation and lipid metabolism, and are also involved in the development of fatty liver disease. In the current study, we investigated the effect of low, but physiologically relevant, concentrations of perfluorooctanoic acid (PFOA), heptafluorobutyric acid (HFBA), and perfluorotetradecanoic acid (PFTA) on gene expression markers of an inflammatory response (tnfa, il-1b, il-6, rplp0, edem1, and dnajc3a), unfolded protein response (UPR) (bip, atf4a, atf6, xbp1, and ddit3), senescence (p21, pai1, smp30, mdm2, and baxa), lipogenesis (scd1, acc, srebp1, pparγ, and fasn) and autophagy (p62, atg3, atg7, rab7, lc3b, and becn1) in AB wild-type (+/+), spns1-wt sibling (+/+), (+/-) and spns1 homozygous mutant (-/-) zebrafish embryos. Exposure to PFOA and HFBA (50 and 100 nM) specifically modulated inflammatory, UPR, senescence, lipogenic, and autophagy signaling in spns1-wt (+/+), (+/-), and spns1-mutant (-/-) zebrafish embryos. Furthermore, PFOA, but not HFBA, upregulated lipogenic-related gene expression and enhanced hepatic steatosis in spns1-wt (+/+), (+/-) zebrafish embryos. Combined exposure to PFOA, HFBA, and PFTA differentially expressed inflammatory, senescence, lipogenic, and autophagy-associated gene expression in spns1-mutant (-/-) zebrafish embryos compared with spns1-wt (+/+), (+/-) and AB-wt (+/+) zebrafish embryos. In addition, chronic exposure (∼2 months) to PFOA (120-600 nM) upregulated the expression of hepatic lipogenic and steatosis biomarkers in AB-wt (+/+) zebrafish. Collectively, our data suggest that acute/chronic physiologically relevant concentrations of PFOA upregulate inflammatory, UPR, senescence, and lipogenic signaling in spns1-wt (+/+), (+/-) and spns1-mutant (-/-) zebrafish embryos as well as in two-month-old AB-wt zebrafish, by targeting autophagy and hence induces toxicity that could promote nonalcoholic fatty liver disease.

Development of an animal model for radiofrequency ablation of primary, virally induced hepatocellular carcinoma in the woodchuck.

PURPOSE: To develop a consistent and reproducible method in an animal model for studies of radiofrequency (RF) ablation of primary hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Fifteen woodchucks were inoculated with woodchuck hepatitis virus (WHV) to establish chronic infections. When serum γ-glutamyl transpeptidase levels became elevated, the animals were evaluated with ultrasound, and, in most cases, preoperative magnetic resonance (MR) imaging to confirm tumor development. Ultimately, RF ablation of tumors was performed by using a 1-cm probe with the animal submerged in a water bath for grounding. Ablation effectiveness was evaluated with contrast-enhanced MR imaging and gross and histopathologic analysis. RESULTS: RF ablation was performed in 15 woodchucks. Modifications were made to the initial study design to adapt methodology for the woodchuck. The last 10 of these animals were treated with a standardized protocol using a 1-cm probe that produced a consistent area of tumor necrosis (mean size of ablation, 10.2 mm × 13.1 mm) and led to no complications. CONCLUSIONS: A safe, reliable and consistent method was developed to study RF ablation of spontaneous primary HCC using chronically WHV-infected woodchucks, an animal model of hepatitis B virus-induced HCC.

Using rheology to quantify the effects of localized collagenase treatments on uterine fibroid digestion.

Uterine fibroids are stiff, benign tumors containing excessive, disordered collagens that occur in 70-80% of women before age 50 and cause bleeding and pain. Collagenase Clostridium histolyticum (CCH) is a bacterial enzyme capable of digesting the collagens present in fibroids. By combining CCH with injectable drug delivery systems to enhance effectiveness, a new class of treatments could be developed to reduce the stiffness of fibroids, preventing the need for surgical removal and preserving fertility. In this work, we achieved localization of CCH via physical entrapment by co-injecting a thermoresponsive pNIPAM-based polymeric delivery system called LiquoGel (LQG), which undergoes a sol-gel transition upon heating. Toxicity study results for LQG injected subcutaneously into mice demonstrate that LQG does not induce lesions or other adverse effects. We then used rheology to quantify the effects of localized CCH injections on the modulus and viscoelasticity of uterine fibroids, which exhibit gel-like behavior, through ex vivo and in vivo digestion studies. Ex vivo CCH injections reduce the tissue modulus by over two orders of magnitude and co-injection of LQG enhances this effect. Rheological results from an in vivo digestion study in mice show a significant reduction in tissue modulus and increase in tissue viscoelasticity 7 days after a single injection of LQG+CCH. Parallel histological staining validates that the observed rheological changes correspond to an increase in collagen lysis after treatment by LQG+CCH. These results show promise for development of injectable and localized enzymatic therapies for uterine fibroids and other dense tumors. STATEMENT OF SIGNIFICANCE: Uterine fibroids are stiff, benign tumors containing high collagen levels that cause bleeding and pain in women. Fertility-preserving and minimally-invasive treatments to soften fibroids are needed as an alternative to surgical removal via hysterectomy. We demonstrate through ex vivo and in vivo studies that co-injecting a thermoresponsive polymer delivery system (LQG) alongside a bacterial collagenase (CCH) enzyme significantly increases treatment effectiveness at softening fibroids through CCH localization. We use rheology to measure the modulus and viscoelasticity of fibroids and histology to show that fibroid softening corresponds to a decrease in collagen after treatment with LQG+CCH. These results highlight the utility of rheology at quantifying tissue properties and present a promising injectable therapy for fibroids and other dense tumors.

Persistent viremia by a novel parvovirus in a slow loris (Nycticebus coucang) with diffuse histiocytic sarcoma.

Cancer is one of the leading health concerns for human and animal health. Since the tumorigenesis process is not completely understood and it is known that some viruses can induce carcinogenesis, it is highly important to identify novel oncoviruses and extensively study underlying oncogenic mechanisms. Here, we investigated a case of diffuse histiocytic sarcoma in a 22 year old slow loris (Nycticebus coucang), using a broad spectrum virus discovery technique. A novel parvovirus was discovered and the phylogenetic analysis performed on its fully sequenced genome demonstrated that it represents the first member of a novel genus. The possible causative correlation between this virus and the malignancy was further investigated and 20 serum and 61 organ samples from 25 animals (N. coucang and N. pygmaeus) were screened for the novel virus but only samples collected from the originally infected animal were positive. The virus was present in all tested organs (intestine, liver, spleen, kidneys, and lungs) and in all banked serum samples collected up to 8 years before death. All attempts to identify a latent viral form (integrated or episomal) were unsuccessful and the increase of variation in the viral sequences during the years was consistent with absence of latency. Since it is well known that parvoviruses are dependent on cell division to successfully replicate, we hypothesized that the virus could have benefitted from the constantly dividing cancer cells and may not have been the cause of the histiocytic sarcoma. It is also possible to conjecture that the virus had a role in delaying the tumor progression and this report might bring new exciting opportunities in recognizing viruses to be used in cancer virotherapy.