Relationship between locomotor activity rhythm and corticosterone levels during HCC development, progression, and treatment in a mouse model.

Cancer-related fatigue (CRF) and stress are common symptoms in cancer patients and represent early side effects of cancer treatment which affect the life quality of the patients. CRF may partly depend on disruption of the circadian rhythm. Locomotor activity and corticosterone rhythms are two important circadian outputs which can be used to analyze possible effects on the circadian function during cancer development and treatment. The present study analyzes the relationship between locomotor activity rhythm, corticosterone levels, hepatocellular carcinoma (HCC) development, and radiotherapy treatment in a mouse model. HCC was induced in mice by single injection of diethylnitrosamine (DEN) and chronic treatment of phenobarbital in drinking water. Another group received chronic phenobarbital treatment only. Tumor bearing animals were divided randomly into four groups irradiated at four different Zeitgeber time points. Spontaneous locomotor activity was recorded continuously; serum corticosterone levels and p-ERK immunoreaction in the suprachiasmatic nucleus (SCN) were investigated. Phenobarbital treated mice showed damped corticosterone levels and a less stable 24 hours activity rhythm as well as an increase in activity during the light phase, reminiscent of sleep disruption. The tumor mice showed an increase in corticosterone level during the inactive phase and decreased activity during the dark phase, reminiscent of CRF. After irradiation, corticosterone levels were further increased and locomotor activity rhythms were disrupted. Lowest corticosterone levels were observed after irradiation during the early light phase; thus, this time might be the best to apply radiotherapy in order to minimize side effects.

Histone chaperone FACT complex mediates oxidative stress response to promote liver cancer progression.

OBJECTIVE: Facilitates Chromatin Transcription (FACT) complex is a histone chaperone participating in DNA repair-related and transcription-related chromatin dynamics. In this study, we investigated its oncogenic functions, underlying mechanisms and therapeutic implications in human hepatocellular carcinoma (HCC). DESIGN: We obtained HCC and its corresponding non-tumorous liver samples from 16 patients and identified FACT complex as the most upregulated histone chaperone by RNA-Seq. We further used CRISPR-based gene activation and knockout systems to demonstrate the functions of FACT complex in HCC growth and metastasis. Functional roles and mechanistic insights of FACT complex in oxidative stress response were investigated by ChIP assay, flow cytometry, gene expression assays and 4sU-DRB transcription elongation assay. Therapeutic effect of FACT complex inhibitor, Curaxin, was tested in both in vitro and in vivo models. RESULTS: We showed that FACT complex was remarkably upregulated in HCC and contributed to HCC progression. Importantly, we unprecedentedly revealed an indispensable role of FACT complex in NRF2-driven oxidative stress response. Oxidative stress prevented NRF2 and FACT complex from KEAP1-mediated protein ubiquitination and degradation. Stabilised NRF2 and FACT complex form a positive feedback loop; NRF2 transcriptionally activates the FACT complex, while FACT complex promotes the transcription elongation of NRF2 and its downstream antioxidant genes through facilitating rapid nucleosome disassembly for the passage of RNA polymerase. Therapeutically, Curaxin effectively suppressed HCC growth and sensitised HCC cell to sorafenib. CONCLUSION: In conclusion, our findings demonstrated that FACT complex is essential for the expeditious HCC oxidative stress response and is a potential therapeutic target for HCC treatment.

Assessing Tumor Mechanics by MR Elastography at Different Strain Levels.

BACKGROUND: Malignant tumors are associated with increased tissue rigidity, which can be an indicator of tumor progression. MR elastography (MRE) has the potential to study the variations of tumor mechanical properties. ex vivo studies have shown the ability of MRE to assess increase of mechanical properties; nevertheless, it has not yet been observed in vivo. PURPOSE: To propose a method to assess the increase in mechanical properties of tumors in vivo under static external compression using MRE. STUDY TYPE: Prospective, experimental study. ANIMAL MODEL: Forty-six SCID mice with subcutaneous tumor implantation (patient-derived hepatocellular carcinoma xenografts, Model 1, n = 13, and Model 2, n = 33). FIELD STRENGTH/SEQUENCE: 7.0T; a spin echo sequence was used for anatomical images and a modified spin echo sequence for elastography acquisitions with a vibration frequency of 600 Hz. ASSESSMENT: An inflatable balloon was placed on the abdomen to apply a load to the tumor. MRE acquisitions were performed at the basal state and at increasing compression levels. Anatomical images were used to calculate the octahedral shear strain between the tumor at the basal strain state and each strain level. For six mice (Model 2), each static preloading scan was acquired twice consecutively without moving the mouse to evaluate repeatability. Statistical Tests: The Bland-Altman method was used to assess repeatability. Correlations between tumor stiffness and deformation were evaluated with Pearson correlation coefficients. RESULTS: For stiffness (G*), a good repeatability was obtained between the acquisitions; the limits of agreement of the Bland-Altman test were [-10.17%; 11.49%] with an absolute bias of 0.66%. A significant correlation between tumor stiffness and deformation was observed for both models (Model 1: r = 0.57, P < 0.0001 and Model 2: r = 0.31, P < 0.0001). DATA CONCLUSION: We establish that tumor mechanical properties can increase under mechanical compression. This increase can effectively be monitored using a proposed MRE setup. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2019;50:1982-1989.

Phytochemicals, antioxidant activity and hepatoprotective effect of ginger (Zingiber officinale) on diethylnitrosamine toxicity in rats.

Context: Chronic liver damage has serious medical consequences. Objective: To investigate the hepatoprotective effect of dry Zingiber officinale (ginger) and its essential (volatile) oil against diethylnitrosamine (DEN) toxicity in rats. Materials and methods: Phenols and flavonoids components were characterized in dry ginger using HPLC-UV instrument while ginger essential oil (E.O.) was investigated via GC-MS technique. Antioxidant activity was determined in vitro. In rat model, ginger was administrated for 2 months. Lipid profile, antioxidant biomarkers, liver functions and histopathology were assessed. Results: Chlorogenic acid (63.85 ppm) and hesperidin (156.91 ppm) are among the major phenolic and flavonoid constituents in dry ginger. Curcumene (15.21%) and linalool (13.47%) represent the main E.O. constituents. In rats treated with ginger E.O., a significant elevation in serum HDL (31.14%) was accompanied by a decrease in LDL (55.14%). A significant decrease in serum ALT and ALP was reported (56.85% and 53.84%, respectively). Serum GSH-Px activity has significantly increased 75.06%. Meanwhile, E.O. showed anticancer potential against HepG2 cell line (IC50 = 40 µg/mL). Liver histopathological examinations confirmed the protective effect against abnormalities. Conclusion: Ginger was able to reduce the severity of DEN-cytotoxicity in rats, which suggests a novel antioxidant role originating from this medicinal plant.

Carvedilol suppresses circulating and hepatic IL-6 responsible for hepatocarcinogenesis of chronically damaged liver in rats.

Carvedilol is an anti-oxidant non-selective ß-blocker used for reduction of portal blood pressure, prophylaxis of esophageal varices development and bleeding in chronic liver diseases. Recently, it exhibited potent anti-inflammatory, anti-fibrotic, anti-proliferative and anti-carcinogenic effects. In the present study, we evaluated the possible suppressive effect of carvedilol on circulating and hepatic IL-6 levels responsible for hepatocarcinogenesis in a rat model of hepatic cirrhosis. Besides, its effect on hepatic STAT-3 levels, function tests, oxidative stress markers, and hydroxyproline content, hepatic tissue histopathological changes and immunohistochemical expression of E & N-cadherin. Nine-week-old male Wistar rats injected intraperitoneal by 1ml/kg 10% CCL4 in olive oil three times/week (every other day) for 12weeks to induce hepatic cirrhosis. Carvedilol (10mg/kg/day suspended in 0.5% CMC orally), silymarin (50mg/kg/day suspended in 0.5% CMC orally) or combination of both used to treat hepatic cirrhosis from 15th to 84th day. Our data showed that carvedilol and silymarin co-treatment each alone or in combination efficiently reduced the elevated serum IL-6, ALT, AST, ALP and BIL, hepatic IL-6, STAT-3, MDA levels and hydroxyproline content. In addition, it elevated the reduced serum ALB level, hepatic CAT activity and GSH level. Meanwhile, it apparently restored the normal hepatic architecture, collagen distribution and immunohistochemical E & N-cadherin expression. Furthermore, carvedilol was superior to silymarin in improving MDA level. Moreover, the combination of carvedilol and silymarin showed an upper hand in amelioration of the CCL4 induced hepatotoxicity than each alone. Therefore, carvedilol could be promising in prevention of hepatocarcinogenesis in chronic hepatic injuries.

Detection and differentiation of early hepatocellular carcinoma from cirrhosis using CT perfusion in a rat liver model.

BACKGROUND: Functional imaging such as CT perfusion can detect morphological and hemodynamic changes in hepatocellular carcinoma (HCC). Pre-carcinoma and early HCC nodules are difficult to differentiate by observing only their hemodynamics changes. The present study aimed to investigate hemodynamic parameters and evaluate their differential diagnostic cut-off between pre-carcinoma and early HCC nodules using CT perfusion and receiver operating characteristic (ROC) curves. METHODS: Male Wistar rats were randomly divided into control (n=20) and experimental (n=70) groups. Diethylnitrosamine (DEN) was used to induce pre-carcinoma and early HCC nodules in the experimental group. Perfusion scanning was carried out on all survival rats discontinuously from 8 to 16 weeks. Hepatic portal perfusion (HPP), hepatic arterial fraction (HAF), hepatic arterial perfusion (HAP), hepatic blood volume (HBV), hepatic blood flow (HBF), mean transit time (MTT) and permeability of capillary vessel surface (PS) data were provided by mathematical deconvolution model. The perfusion parameters were compared among the three groups of rats (control, pre-carcinoma and early HCC groups) using the Kruskal-Wallis test and analyzed with ROC curves. Histological examination of the liver tissues with hematoxylin and eosin staining was performed after CT scan. RESULTS: For HPP, HAF, HBV, HBF and MTT, there were significant differences among the three groups (P<0.05). HAF had the highest areas under the ROC curves: 0.80 (control vs pre-carcinoma groups) and 0.95 (control vs early HCC groups) with corresponding optimal cut-offs of 0.37 and 0.42, respectively. The areas under the ROC curves for HPP was 0.79 (control vs pre-carcinoma groups) and 0.92 (control vs early HCC groups) with corresponding optimal cut-offs of 136.60 mL/min/100 mg and 108.47 mL/min/100 mg, respectively. CONCLUSIONS: CT perfusion combined with ROC curve analysis is a new diagnosis model for distinguishing between pre-carcinoma and early HCC nodules. HAF and HPP are the ideal reference indices.

Regular exercise decreases liver tumors development in hepatocyte-specific PTEN-deficient mice independently of steatosis.

BACKGROUND & AIMS: Unhealthy lifestyles predispose people to non-alcoholic steatohepatitis (NASH), which may further result in the development of hepatocellular carcinoma (HCC). Although NASH patients benefit from physical activity, it is unknown whether regular exercise reduces the risk of developing HCC. Therefore, we studied the effect of regular exercise on the development of HCC in male hepatocyte-specific PTEN-deficient mice (AlbCrePten(flox/flox)), which develop steatohepatitis and HCC spontaneously. METHODS: Mice were fed a standardized 10% fat diet and were randomly divided into exercise or sedentary groups. The exercise group ran on a motorized treadmill for 60 min/day, 5 days/week during 32 weeks. RESULTS: After 32 weeks of regular exercise, 71% of exercised mice developed nodules larger than 15 mm(3)vs. 100% of mice in the sedentary group. The mean number of tumors per liver was reduced by exercise, as well as the total tumoral volume per liver. Exercise did not affect steatosis and had no effect on the non-alcoholic fatty liver disease (NAFLD) Activity Score (NAS). Exercise decreased tumor cell proliferation. Mechanistically, exercise stimulated the phosphorylation of AMPK and its substrate raptor, which decreased the kinase activity of mTOR. CONCLUSIONS: These data show a beneficial effect of regular exercise on the development of HCC in an experimental model of NASH and offer a rationale for encouraging predisposed patients to increase their physical activity for the prevention of HCC.

p28(GANK) prevents degradation of Oct4 and promotes expansion of tumor-initiating cells in hepatocarcinogenesis.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is believed to arise from tumor-initiating cells (T-ICs), although little is known about their stem cell-like properties. METHODS: We quantified levels of p28(GANK) (Gankyrin), OV6, and Oct4 in 130 human HCC samples using immunohistochemistry. Magnetic-activated cell sorting was used to isolate OV6+ HCC cells. T-IC properties were evaluated by quantitative reverse-transcription polymerase chain reaction, flow cytometry, and spheroid formation. We used a coimmunoprecipitation assay to study interactions among p28(GANK), Oct4, and WWP2. Tumorigenicity and pulmonary metastasis were examined in nonobese diabetic and severe combined immunodeficient mice. RESULTS: In HCC samples, high levels of p28(GANK) correlated with expansion of OV6+ tumor cells; the combination of high levels of p28(GANK) and OV6 was associated with progression of HCC. p28(GANK) was predominantly expressed in liver T-ICs, isolated by magnetic sorting, and undifferentiated primary HCC spheroids. Increased levels of p28(GANK) in T-ICs increased their percentages in HCC samples, expression of stem cell genes, self-renewal potential, chemoresistance in vitro, and tumorigenicity and ability to develop into pulmonary metastases in mice. Conversely, knockdown of p28(GANK) reduced their T-IC properties. p28(GANK) likely activates liver T-ICs by impeding ubiquitination and degradation of the transcription factor Oct4 by WWP2. In support of this concept, levels of p28(GANK) correlated with those of Oct4 in HCC samples. CONCLUSIONS: p28(GANK) activates and maintains liver T-ICs in HCCs by preventing degradation of Oct4. Inhibitors of p28(GANK) might therefore be developed to inactivate T-ICs and slow tumor progression.

MicroRNA-194 is a target of transcription factor 1 (Tcf1, HNF1α) in adult liver and controls expression of frizzled-6.

UNLABELLED: Transcription factor 1 (Tcf1; hepatocyte nuclear factor 1α [HNF1α]) is critical for hepatocyte development and function. Whether Tcf1 also regulates hepatic microRNAs (miRNAs) has not been investigated yet. Here we analyzed Tcf1-dependent miRNA expression in adult mice in which this transcription factor had been genetically deleted (Tcf1(-/-) ) using miRNA microarray analysis. The miR-192/-194 cluster was markedly down-regulated in liver of Tcf1(-/-) mice. MiR-192/-194 levels were also decreased in two other tissues that express Tcf1, kidney and small intestine, although to a lesser extent than in liver. In order to identify targets of miR-192/-194 in vivo we combined Affymetrix gene analysis of liver in which miR-192/-194 had been silenced or overexpressed, respectively, and tested regulated messenger RNAs (mRNAs) with multiple binding sites for these miRNAs. This approach revealed frizzled-6 (Fzd6) as a robust endogenous target of miR-194. MiR-194 also targets human FZD6 and expression of miR-194 and Fzd6 are inversely correlated in a mouse model of hepatocellular carcinoma (Dgcr8(flox/flox) p53(flox/flox) × Alb-Cre). CONCLUSION: Our results support a role of miR-194 in liver tumorigenesis through its endogenous target Fzd6. These results may have important implications for Tcf1-mediated liver proliferation.

Hepatitis B virus X (HBx) induces tumorigenicity of hepatic progenitor cells in 3,5-diethoxycarbonyl-1,4-dihydrocollidine-treated HBx transgenic mice.

UNLABELLED: Hepatitis B virus X (HBx) protein is implicated in hepatitis B virus (HBV)-associated liver carcinogenesis. However, it remains unclear whether HBx-expressing hepatic progenitor cells (HPCs) are attributed to liver tumor formation. In this study, by using HBx transgenic mice and a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced liver injury model, the relationship between HBx expression and tumorigenicity of HPCs was analyzed. Compared with control mice, an elevated number of EpCAM(+) cells with characteristics of HPCs was observed in HBx mice after 1 month and 4 months of DDC diet feeding. All HBx transgenic mice developed liver tumors characterized by histological features of both hepatocellular carcinoma (HCC) and cholangiocarcinoma after 7 months of DDC feeding. Notably, EpCAM(+) HPCs isolated from premalignant HBx mice exposed to a DDC diet for 4 months formed subcutaneous mixed-lineage tumors (four out of six) in nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice, and none of the cells from wildtype (WT) induced tumor, indicating that HBx may induce malignant transformation of HPCs that contributes to tumorigenesis. We also found higher titers of circulating interleukin (IL)-6, activities of IL-6/STAT3, and Wnt/ß-catenin signaling pathways in HBx transgenic mice, suggesting HBx may induce intrinsic changes in HPCs by way of the above signaling that enables HPCs with tumorigenicity potential. Finally, clinical evidence showed that high HBx expression in human HBV-related HCC was statistically associated with expansion of EpCAM(+) or OV6(+) tumor cells and aggressive clinicopathologic features. CONCLUSION: HBx induces intrinsic cellular transformation promoting the expansion and tumorigenicity of HPCs in DDC-treated mice, which may be a possible origin for liver cancer induced by chronic hepatitis infection.

Gastrodin stimulates anticancer immune response and represses transplanted H22 hepatic ascitic tumor cell growth: Involvement of NF-κB signaling activation in CD4+ T cells.

Gastrodia elata Blume (G. elata) is a famous restorative food in East Asia. It can be used as an auxiliary reagent in hepatocellular carcinoma (HCC) treatment. Previous studies unveiled that G. elata exhibited immunomodulatory activities. To explore the active ingredients contributing to its immunomodulatory activities, gastrodin, vanillin, and parishin B were purified from G. elata and their anti-HCC effects were assessed in vivo. Among these compounds, only gastrodin was capable of repressing transplanted H22 ascitic hepatic tumor cell growth in vivo with low toxicity. Further investigations were designed to explore the effects of gastrodin on the immune system of tumor-bearing mice and potential molecular mechanisms underlying these effects. Our data showed that gastrodin ameliorated tumor cell transplantation-induced activation of endogenous pro-apoptotic pathway in CD4+ T cells and abnormalities in serum cytokine profiles in host animals. These events enhanced cytotoxic activities of natural killer and CD8+ T cells against H22 hepatic cancer cells. Gastrodin administration specifically upregulated mRNA levels of several nuclear factor κB (NF-κB) responsive genes in CD4+ T cells but not in CD8+ T cells. Chromatin immunoprecipitation assay showed that gastrodin increased the association of NF-κB p65 subunit to the promoter regions of IL-2 and Bcl-2 encoding genes in CD4+ T cells. Our investigations demonstrated that gastrodin is the main active ingredient contributing to the anticancer immunomodulatory properties of G. elata. Promoting NF-κB-mediated gene transcription in CD4+ T cells is implicated in its immunomodulatory activity.

Diet-induced obesity and ethanol impair progression of hepatocellular carcinoma in a mouse mesenteric vein injection model.

BACKGROUND: Hepatocellular carcinoma (HCC) is a rapidly increasing cancer whose known risk factors are chronic ethanol abuse, viral hepatitis infection, and aflatoxin exposure. Obesity, an emerging HCC risk factor, is reaching epidemic proportions in developed nations. This study investigated the effects of diet-induced obesity (DIO) and chronic ethanol consumption on HCC progression in mice in vivo. METHODS: In this study, C57BL/6 DIO mice and lean litter mates were maintained on a 60% (high-fat diet [HFD]) diet or a 10% (control diet [CD]) kcal% fat diet for 7 weeks before they were weaned to 10/20% ([v/v], alternating days) ethanol in drinking water (EtOH) or maintenance on drinking water (H(2)O) alone. Hepatic tumor formation was initiated by intrahepatic Hepa1-6 cell (6 × 10(6) cells) inoculation 6 weeks later via the mesenteric vein. RESULTS: The animals receiving the HFD showed decreased tumor incidence and area of hepatic foci versus the CD animals maintained on H(2)O alone. The action of EtOH suppressed tumor incidence further in both the CD and the HFD mice. Serologic analysis showed no significant differences in liver enzymes among the groups. Protein analysis demonstrated increased P450 2E1 (CYP2E1) in the groups maintained on EtOH, an effect exacerbated by HFD. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated increased tumor necrosis factor-alpha (TNF-α) expression in HFD HCC mice (H(2)O and EtOH) concomitant with decreased transforming growth factor-beta (TGF-ß) expression. CONCLUSIONS: Although obesity and EtOH consumption are known risk factors for HCC initiation and development, the data in this study suggest that these factors impair progression of established tumors within the liver.

Accelerated carcinogenesis following liver regeneration is associated with chronic inflammation-induced double-strand DNA breaks.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide and is considered to be the outcome of chronic liver inflammation. Currently, the main treatment for HCC is surgical resection. However, survival rates are suboptimal partially because of tumor recurrence in the remaining liver. Our aim was to understand the molecular mechanisms linking liver regeneration under chronic inflammation to hepatic tumorigenesis. Mdr2-KO mice, a model of inflammation-associated cancer, underwent partial hepatectomy (PHx), which led to enhanced hepatocarcinogenesis. Moreover, liver regeneration in these mice was severely attenuated. We demonstrate the activation of the DNA damage-response machinery and increased genomic instability during early liver inflammatory stages resulting in hepatocyte apoptosis, cell-cycle arrest, and senescence and suggest their involvement in tumor growth acceleration subsequent to PHx. We propose that under the regenerative proliferative stress induced by liver resection, the genomic unstable hepatocytes generated during chronic inflammation escape senescence and apoptosis and reenter the cell cycle, triggering the enhanced tumorigenesis. Thus, we clarify the immediate and long-term contributions of the DNA damage response to HCC development and recurrence.

Residual dormant cancer stem-cell foci are responsible for tumor relapse after antiangiogenic metronomic therapy in hepatocellular carcinoma xenografts.

Hepatocellular carcinoma (HCC) is the fifth most common solid tumor and the third leading cause of cancer-related deaths. Currently available chemotherapeutic options are not curative due in part to tumor resistance to conventional therapies. We generated orthotopic HCC mouse models in immunodeficient NOD/SCID/IL2rγ null mice by injection of human alpha-feto protein (hAFP)- and/or luciferase-expressing HCC cell lines and primary cells from patients, where tumor growth and spread can be accurately monitored in a non-invasive way. In this model, low-dose metronomic administration of cyclophosphamide (LDM-CTX) caused complete regression of the tumor mass. A significant increase in survival (P<0.0001), reduced aberrant angiogenesis and hyperproliferation, and decrease in the number of circulating tumor cells were found in LDM-CTX-treated animals, in comparison with untreated mice. Co-administration of LDM-CTX with anti-VEGF therapy further improved the therapeutic efficacy. However, the presence of residual circulating hAFP levels suggested that some tumor cells were still present in livers of treated mice. Immunohistochemistry revealed that those cells had a hAFP+/CD13+/PCNA- phenotype, suggesting that they were dormant cancer stem cells (CSC). Indeed, discontinuation of therapy resulted in tumor regrowth. Moreover, in-vitro LDM-CTX treatment reduced hepatosphere formation in both number and size, and the resulting spheres were enriched in CD13+ cells indicating that these cells were particularly resistant to therapy. Co-treatment of the CD13-targeting drug, bestatin, with LDM-CTX leads to slower tumor growth and a decreased tumor volume. Therefore, combining a CD13 inhibitor, which targets the CSC-like population, with LDM-CTX chemotherapy may be used to eradicate minimal residual disease and improve the treatment of liver cancer.

Differential expression of TRPM7 in rat hepatoma and embryonic and adult hepatocytes.

TRPM7 channels are implicated in cellular survival, proliferation, and differentiation. However, a profile of TRPM7 activity in a specific cell type has not been determined from embryonic to terminally differentiated state. Here, we characterized TRPM7 expression in a spectrum of rat liver cells at different developmental stages. Using the whole-cell patch clamp technique, TRPM7-like Na(+) currents were identified in RLC-18 cells, a differentiated, proliferating hepatocellular line derived from day 17 embryonic rat liver. Currents were outwardly rectifying, enhanced in divalent-free solutions, and inhibited by intracellular Mg(2+). Reverse transcription - polymerase chain reaction (RT-PCR) revealed that RLC-18 cells express both TRPM6 and TRPM7. However, mean currents were reduced almost 80% by 1 mmol/L 2-aminoethoxyphenylborate (2-APB) and were abolished in RLC-18 cells heterologously expressing a dominant negative TRPM7 construct, suggesting that TRPM7 is the major current carrier in these cells. Functional comparison showed that relative to terminally differentiated adult rat hepatocytes, currents were 1.8 and 3.9 times higher in, respectively, RLC-18 and WIF-B cells, a rat hepatoma - human fibroblast cross. Our results demonstrate that plasma membrane TRPM7 channels are more highly expressed in proliferating cells as compared with terminally differentiated and nondividing rat hepatocytes and suggest that downregulation of this channel is associated with hepatocellular differentiation.

Toll-like receptor 4 mediates synergism between alcohol and HCV in hepatic oncogenesis involving stem cell marker Nanog.

Alcohol synergistically enhances the progression of liver disease and the risk for liver cancer caused by hepatitis C virus (HCV). However, the molecular mechanism of this synergy remains unclear. Here, we provide the first evidence that Toll-like receptor 4 (TLR4) is induced by hepatocyte-specific transgenic (Tg) expression of the HCV nonstructural protein NS5A, and this induction mediates synergistic liver damage and tumor formation by alcohol-induced endotoxemia. We also identify Nanog, the stem/progenitor cell marker, as a novel downstream gene up-regulated by TLR4 activation and the presence of CD133/Nanog-positive cells in liver tumors of alcohol-fed NS5A Tg mice. Transplantation of p53-deficient hepatic progenitor cells transduced with TLR4 results in liver tumor development in mice following repetitive LPS injection, but concomitant transduction of Nanog short-hairpin RNA abrogates this outcome. Taken together, our study demonstrates a TLR4-dependent mechanism of synergistic liver disease by HCV and alcohol and an obligatory role for Nanog, a TLR4 downstream gene, in HCV-induced liver oncogenesis enhanced by alcohol.

Synergism between p53 and Mcl-1 in protecting from hepatic injury, fibrosis and cancer.

BACKGROUND & AIMS: Mcl-1-deficient hepatocytes are prone to undergo apoptosis. The tumor suppressor protein p53 plays an important role in apoptosis control as well as other cellular responses. This study was initially aimed to examine whether p53 was involved in Mcl-1 deficiency-induced apoptosis of hepatocytes. METHODS: Hepatocyte-specific Mcl-1 knockout (Alb-Mcl-1(-/-)) mice and Alb-Mcl-1(-/-) mice in wild-type or p53-deficient background were generated and characterized. RESULTS: Alb-Mcl-1(-/-) mice were viable, but their liver cells were prone to undergo apoptosis and manifested a slightly elevated level of p53. To examine the role of p53 in Alb-Mcl-1(-/-) livers, Alb-Mcl-1(-/-) mice without p53 (DKO mice) were characterized. Unexpectedly, although p53-deficient mice appeared to be developmentally normal, DKO mice were highly susceptible to neonatal death (∼60%). Further analysis revealed that such an early lethality was likely due to hepatic failure caused by a marked reduction of fully-differentiated hepatocytes at the perinatal/neonatal stage. Moreover, those DKO mice that did survive to adulthood manifested more severe liver damage than Alb-Mcl-1(-/-) mice, suggesting that p53 was activated in Alb-Mcl-1(-/-) livers to promote cell survival. Microarray followed by quantitative PCR analysis suggested that p21(Waf1/Cip1), one p53 target gene with apoptosis-inhibitory function, is likely involved in the protective role of p53 in Alb-Mcl-1(-/-) livers. Moreover, we demonstrated that loss of p53 promoted liver fibrosis and tumor development in Alb-Mcl-1(-/-) mice. CONCLUSIONS: This study revealed an unexpected synergism between Mcl-1 and p53 in protecting from hepatic injury, fibrosis, and cancer.

Selective targeting of genetically engineered mesenchymal stem cells to tumor stroma microenvironments using tissue-specific suicide gene expression suppresses growth of hepatocellular carcinoma.

BACKGROUND: The use of engineered mesenchymal stem cells (MSCs) as therapeutic vehicles for the treatment of experimental pancreatic and breast cancer has been previously demonstrated. The potential application of MSCs for the treatment of hepatocellular carcinoma (HCC) has been controversial. The general approach uses engineered MSCs to target different aspects of tumor biology, including angiogenesis or the fibroblast-like stromal compartment, through the use of tissue-specific expression of therapeutic transgenes. The aim of the present study was (1) to evaluate the effect of exogenously added MSCs on the growth of HCC and (2) the establishment of an MSC-based suicide gene therapy for experimental HCC. METHODS: Mesenchymal stem cells were isolated from bone marrow of C57/Bl6 p53(-/-) mice. The cells were injected into mice with HCC xenografts and the effect on tumor proliferation and angiogenesis was evaluated. The cells were then stably transfected with red fluorescent protein (RFP) or Herpes simplex virus thymidine kinase (HSV-Tk) gene under control of the Tie2 promoter/enhancer or the CCL5 promoter. Mesenchymal stem cells were injected intravenously into mice with orthotopically growing xenografts of HCC and treated with ganciclovir (GCV). RESULTS: Ex vivo examination of hepatic tumors revealed tumor-specific recruitment, enhanced tumor growth, and increased microvessel density after nontherapeutic MSC injections. After their homing to the hepatic xenografts, engineered MSCs demonstrated activation of the Tie2 or CCL5 promoter as shown by RFP expression. Application of CCL5/HSV-TK transfected MSCs in combination with GCV significantly reduced tumor growth by 56.4% as compared with the control group and by 71.6% as compared with nontherapeutic MSC injections. CCL5/HSV-TK(+) transfected MSCs proved more potent in tumor inhibition as compared with Tie2/HSV-TK(+) MSCs. CONCLUSION: Exogenously added MSCs are recruited to growing HCC xenografts with concomitant activation of the CCL5 or Tie2 promoters within the MSCs. Stem cell-mediated introduction of suicide genes into the tumor followed by prodrug administration was effective for treatment of experimental HCC and thus may help fill the existing gap in bridging therapies for patients suffering from advanced HCCs.

Perfusion CT assessment of tissue hemodynamics following hepatic arterial infusion of increasing doses of angiotensin II in a rabbit liver tumor model.

PURPOSE: To investigate the effects of increasing doses of angiotensin II on hepatic hemodynamics in the normal rabbit liver and in hepatic VX2 tumors by using dynamic contrast material-enhanced perfusion computed tomography (CT). MATERIALS AND METHODS: This study was approved by the institutional animal care and use committee. Solitary hepatic VX2 tumors were implanted into 12 rabbits. In each animal, perfusion CT of the liver was performed before (at baseline) and after hepatic arterial infusion of varying doses (0.1-50.0 µg/mL) of angiotensin II. Images were acquired continuously for 80 seconds after the start of the intravenous contrast material administration. Blood flow (BF), blood volume (BV), mean transit time (MTT), and capillary permeability-surface area product were calculated for the tumor and the adjacent and distant normal liver tissue. Generalized linear mixed models were used to estimate the effects of angiotensin II dose on outcome measures. RESULTS: Angiotensin II infusion increased contrast enhancement of the tumor and distal liver vessels. Tumor BF increased in a dose-dependent manner after administration of 0.5-25.0 µg/mL angiotensin II, but only the 2.5 µg/mL dose induced a significant increase in tumor BF compared with BF in the adjacent (68.0 vs 26.3 mL/min/100 g, P < .0001) and distant (68.0 vs 28.3 mL/min/100 g, P = .02) normal liver tissue. Tumor BV varied with angiotensin II dose but was greater than the BV of the adjacent and distant liver tissue at only the 2.5 µg/mL (4.8 vs 3.5 mL/100 g for adjacent liver [P < .0001], 4.8 vs 3.3 mL/100 g for distant liver [P = .0006]) and 10.0 µg/mL (4.9 vs 4.4 mL/100 g for adjacent liver [P = .007], 4.9 vs 4.3 mL/100 g for distant liver [P = .04]) doses. Tumor MTT was significantly shorter than the adjacent liver tissue MTT at angiotensin II doses of 2.5 µg/mL (9.7 vs 15.8 sec, P = .001) and 10.0 µg/mL (5.1 vs 13.2 sec, P = .007) and significantly shorter than the distant liver tissue MTT at 2.5 µg/mL only (9.7 vs 15.3 sec, P = .0006). The capillary permeability-surface area product for the tumor was higher than that for the adjacent liver tissue at the 2.5 µg/mL angiotensin II dose only (11.5 vs 8.1 mL/min/100 g, P = .01). CONCLUSION: Perfusion CT enables a mechanistic understanding of angiotensin II infusion in the liver and derivation of the optimal effective dose. The 2.5 µg/mL angiotensin II dose increases perfusion in hepatic VX2 tumors versus that in adjacent and distant normal liver tissue primarily by constricting normal distal liver vessels and in turn increasing tumor BF and BV.

The mechanisms of renin-angiotensin system in hepatocellular carcinoma: From the perspective of liver fibrosis, HCC cell proliferation, metastasis and angiogenesis, and corresponding protection measures.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, of which the occurrence and development involve a variety of pathophysiological processes, such as liver fibrosis, hepatocellular malignant proliferation, metastasis, and tumor angiogenesis. Some important cytokines, such as TGF-ß, PI3K, protein kinase B (Akt), VEGF and NF-κB, can regulate the growth, proliferation, diffusion, metastasis, and apoptosis of HCC cells by acting on the corresponding signaling pathways. Besides, many studies have shown that the formation of HCC is closely related to the main components of renin-angiotensin system (RAS), such as Ang II, ACE, ACE2, MasR, AT1R, and AT2R. Therefore, this review focused on liver fibrosis, HCC cell proliferation, metastasis, tumor angiogenesis, and corresponding protective measures. ACE-Ang II-AT1 axis and ACE2-Ang-(1-7)-MasR axis were taken as the main lines to introduce the mechanism of RAS in the occurrence and development of HCC, so as to provide references for future clinical work and scientific research.