Hepatic gene expression in mouse models of non-alcoholic fatty liver disease after acute exercise.

AIM: Non-alcoholic fatty liver disease (NAFLD) patients benefit from physical exercise. This study aimed to investigate the effect of acute exercise on hepatic gene expression in different mouse models of NAFLD. METHODS: C57BL/6J mice were fed with a control (CD) or a high fat (HFD) diet. AlbCrePtenflox/flox (Pten-KO) and Fxr-/- mice, two genetic models of NAFLD with insulin hypersensitivity and resistance, respectively, were fed with CD. After 4 weeks, mice were randomly assigned to exercise or sedentariness. Mice were killed 15 min or 3 h after the running/sedentary period. Genome-wide hepatic gene expression was evaluated with the Illumina Micro-array platform. Quantitative polymerase chain reaction confirmed changes in gene expression. RESULTS: Acute exercise transiently affected the expression of genes involved in the immune response in C57BL/6 mice fed with CD and this effect normalized in the recovery phase. Acute exercise affected genes involved in gluconeogenesis in the insulin resistant Fxr-/- model. Genes involved in lipid metabolism were affected in C57BL/6 mice fed with CD, but not in mouse models of NAFLD. Genes involved in DNA damage response pathways were deregulated only in C57BL/6 mice fed with CD and not in mouse models of NAFLD. CONCLUSION: The simultaneous analysis of different NAFLD models revealed that an acute exercise bout affects hepatic gene expression differentially according to animal models and that most of the differentially expressed genes are involved in glucose and fatty acid metabolism, immune regulation, and DNA damage response.

PTEN Down-Regulation Promotes ß-Oxidation to Fuel Hypertrophic Liver Growth After Hepatectomy in Mice.

In regenerating liver, hepatocytes accumulate lipids before the major wave of parenchymal growth. This transient, regeneration-associated steatosis (TRAS) is required for liver recovery, but its purpose is unclear. The tumor suppressor phosphatase and tensin homolog (PTEN) is a key inhibitor of the protein kinase B/mammalian target of rapamycin axis that regulates growth and metabolic adaptations after hepatectomy. In quiescent liver, PTEN causes pathological steatosis when lost, whereas its role in regenerating liver remains unknown. Here, we show that PTEN down-regulation promotes liver growth in a TRAS-dependent way. In wild-type mice, PTEN reduction occurred after TRAS formation, persisted during its disappearance, and correlated with up-regulated ß-oxidation at the expense of lipogenesis. Pharmacological modulation revealed an association of PTEN with TRAS turnover and hypertrophic liver growth. In liver-specific Pten-/- mice shortly after induction of knockout, hypertrophic regeneration was accelerated and led to hepatomegaly. The resulting surplus liver mass was functional, as demonstrated by raised survival in a lethal model of resection-induced liver failure. Indirect calorimetry revealed lipid oxidation as the primary energy source early after hepatectomy. The shift from glucose to lipid usage was pronounced in Pten-/- mice and correlated with the disappearance of TRAS. Partial inhibition of ß-oxidation led to persisting TRAS in Pten-/- mice and abrogated hypertrophic liver growth. PTEN down-regulation may promote ß-oxidation through ß-catenin, whereas hypertrophy was dependent on mammalian target of rapamycin complex 1. CONCLUSION: PTEN down-regulation after hepatectomy promotes the burning of TRAS-derived lipids to fuel hypertrophic liver regeneration. Therefore, the anabolic function of PTEN deficiency in resting liver is transformed into catabolic activities upon tissue loss. These findings portray PTEN as a node coordinating liver growth with its energy demands and emphasize the need of lipids for regeneration. (Hepatology 2017;66:908-921).

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.

Virus-like particle-mediated intracellular delivery of mRNA cap analog with in vivo activity against hepatocellular carcinoma.

Adenovirus dodecahedron (Dd), a nanoparticulate proteinaceous biodegradable virus-like particle (VLP), was used as a vector for delivery of an oncogene inhibitor to hepatocellular carcinoma (HCC) rat orthotopic model. Initiation factor eIF4E is an oncogene with elevated expression in human cancers. Cell-impermeant eIF4E inhibitor, cap structure analog (cap) and anti-cancer antibiotic doxorubicin (Dox) were delivered as Dd conjugates. Dd-cap and Dd-dox inhibited cancer cell culture proliferation up to 50 and 84%, respectively, while with free Dox similar results could be obtained only at a 5 times higher concentration. In animal HCC model the combination treatment of Dd-cap/Dd-dox caused 40% inhibition of tumor growth. Importantly, the level of two pro-oncogenes, eIF4E and c-myc, was significantly diminished in tumor sections of treated rats. Attachment to Dd, a virus-like particle, permitted the first demonstration of cap analog intracellular delivery and resulted in improved doxorubicin delivery leading to statistically significant inhibition of HCC tumor growth.

Everolimus is a potent inhibitor of activated hepatic stellate cell functions in vitro and in vivo, while demonstrating anti-angiogenic activities.

Progression of liver fibrosis to HCC (hepatocellular carcinoma) is a very complex process which involves several pathological phenomena, including hepatic stellate cell activation, inflammation, fibrosis and angiogenesis. Therefore inhibiting multiple pathological processes using a single drug can be an effective choice to curb the progression of HCC. In the present study, we used the mTOR inhibitor everolimus to observe its effect on the in vitro activation of hepatic stellate cells and angiogenesis. The results of the present study demonstrated that everolimus treatment blocked the functions of the immortalized human activated hepatic stellate cell line LX-2 without affecting the viability and migration of primary human stellate cells. We also observed that treatment with everolimus (20 nM) inhibited collagen production by activated stellate cells, as well as cell contraction. Everolimus treatment was also able to attenuate the activation of primary stellate cells to their activated form. Angiogenesis studies showed that everolimus blocked angiogenesis in a rat aortic ring assay and inhibited the tube formation and migration of liver sinusoidal endothelial cells. Finally, everolimus treatment reduced the load of tumoral myofibroblasts in a rat model of HCC. These data suggest that everolimus targets multiple mechanisms, making it a potent blocker of the progression of HCC from liver fibrosis.

Radiofrequency ablation suppresses distant tumour growth in a novel rat model of multifocal hepatocellular carcinoma.

RFA (radiofrequency ablation) is an established therapy for HCC (hepatocellular carcinoma). The multikinase inhibitor sorafenib prolongs survival in advanced HCC. We examined the effects of RFA alone and in combination with sorafenib on a bystanding tumour in a two-tumour rat model of HCC. A total of 80 rats were implanted with two liver tumours and randomized to four treatment groups: vehicle and sham operation (control), sorafenib and sham operation (Sora/Sham), vehicle and RFA (Vh/RFA), and sorafenib and RFA (Sora/RFA) (n=10/group per time point). RFA or sham-operation was performed on the left lobe tumour on day 15. Animals were killed at day 18 and day 30. Non-RFA-targeted right lobe tumours were analysed for angiogenesis, growth factors [HGF (hepatocyte growth factor), EGF (epidermal growth factor) and VEGF (vascular endothelial growth factor)] and infiltrating immune cells (CD3 and CD68). At day 30, the non-RFA-targeted tumours were significantly smaller in all three treatment groups compared with control (Sora/Sham P≤0.0001, Vh/RFA P=0.005 and Sora/RFA P≤0.0001). The smallest tumours were observed in animals treated with a combination of sorafenib and RFA, whereas the size reduction seen in the RFA-only group indicated an RFA-mediated distant suppression of tumour growth. Growth factor measurement revealed transiently decreased EGF levels after RFA (P=0.008), whereas sorafenib treatment decreased HGF levels (P=0.001). MVD (microvessel density) was reduced by sorafenib (P=0.002) despite increased VEGF levels (P≤0.0001). The immune parameters revealed augmented T-cells and IL-10 (interleukin 10) levels in all three treatment groups; sorafenib additionally increased macrophage numbers (P≤0.0001). RFA and sorafenib alone resulted in significant volume reduction of the non-RFA-targeted tumour; this effect was enhanced when both modalities were combined.

Erlotinib and sorafenib in an orthotopic rat model of hepatocellular carcinoma.

BACKGROUND & AIMS: The combination of erlotinib with sorafenib is currently being investigated in a phase III RCT. We studied the effect of erlotinib and sorafenib on HCC in a preclinical model. METHODS: The Morris Hepatoma (MH) and HepG2 cells were treated in vitro with sorafenib (1-10 µM) and erlotinib (1-5 µM) and evaluated for tumor cell viability, apoptosis, and target regulation. Antiangiogenic effects were studied by measuring VEGF levels, endothelial cell viability, apoptosis, migration, and the aortic ring assay. In vivo, MH cells were implanted into the liver of syngeneic rats and treated with vehicle, sorafenib 5-10mg/kg, erlotinib 10mg/kg, and respective combinations. RESULTS: In vitro, erlotinib downregulated p-ERK but showed no significant effect on tumor cell viability in MH and HEPG2 cells. Despite a similar target regulation, sorafenib significantly reduced cell viability of HCC cells by induction of apoptosis, in a dose-dependent manner (11 ± 5%; 20 ± 10%; 51 ± 5% for sorafenib 1, 5, 10 µM). No additional effect was observed upon combination with erlotinib. Of note, erlotinib treatment resulted in endothelial cell migration and vascular sprouting of aortic rings through induction of VEGF mRNA and protein levels in endothelial and tumor cells, which was blocked by sorafenib. In vivo, erlotinib had no single agent antitumor activity, raised serum-VEGF levels, and lacked a synergistic effect in combination with sorafenib. CONCLUSIONS: Erlotinib had no antitumor effect on HCC in vitro nor in vivo, but induced VEGF, which may reflect a resistance mechanism to erlotinib monotherapy. No improvement of sorafenib efficacy was observed upon combination with erlotinib.

Progression-Free and Overall Survival for Concurrent Nivolumab With Standard Concurrent Chemoradiotherapy in Locally Advanced Stage IIIA-B NSCLC: Results From the European Thoracic Oncology Platform NICOLAS Phase II Trial (European Thoracic Oncology Platform 6-14).

INTRODUCTION: The NICOLAS study is the first completed single-arm phase II trial in stage III NSCLC evaluating hierarchically first the safety and then the efficacy of adding nivolumab concurrently to standard definitive concurrent chemoradiotherapy. The safety end point was reported earlier; here, we present the efficacy results. METHODS: Stage IIIA-B unresectable treatment-naive patients with NSCLC received three cycles of platinum-based chemotherapy and concurrent radiotherapy (66 Gy, 33 fractions), along with nivolumab (360 mg, 3-weekly). Nivolumab was continued as monotherapy consolidation for a maximum of 1 year (480 mg, 4-weekly). The primary end point was 1-year progression-free survival (PFS), with a target improvement compared with historical data of at least 15%, from 45% to 60%. To test this efficacy hypothesis, a sample size of 74 assessable patients provided a power of 83% with a one-sided alpha of 5%. RESULTS: A total of 79 patients were enrolled with a median follow-up of 21.0 months (interquartile range: 15.8-25.8 mo) for the primary PFS analysis. A total of 35.4% of the patients had stage IIIA, and 63.3% had stage IIIB disease. The 1-year PFS was 53.7% (95% confidence interval [CI]: 42.0%-64.0%) and the median PFS was 12.7 months (95% CI: 10.1-22.8 mo). Because 37 PFS events occurred in the first year posttreatment among the first 74 assessable patients, a 1-year PFS rate of at least 45% could not be rejected (p = 0.23). At an extended follow-up (median 32.6 mo), 37 deaths have been recorded, with a median overall survival (OS) of 38.8 months (95% CI: 26.8 mo-not estimable) and a 2-year OS rate of 63.7% (95% CI: 51.9%-73.4%). The OS of patients with stage IIIA disease was found to be significantly higher than patients with stage IIIB disease, with a 2-year OS of 81% and 56%, respectively (p = 0.037). CONCLUSIONS: PFS and OS are arithmetically higher in studies involving the same population. However, on the basis of the formal hierarchical efficacy analysis, we could not reject that the 1-year PFS rate is at least 45%.

Hypoxia aggravates non-alcoholic steatohepatitis in mice lacking hepatocellular PTEN.

The metabolic disorders that predispose patients to NASH (non-alcoholic steatohepatitis) include insulin resistance and obesity. Repeated hypoxic events, such as occur in obstructive sleep apnoea syndrome, have been designated as a risk factor in the progression of liver disease in such patients, but the mechanism is unclear, in particular the role of hypoxia. Therefore we studied the influence of hypoxia on the development and progression of steatohepatitis in an experimental mouse model. Mice with a hepatocellular-specific deficiency in the Pten (phosphatase and tensin homologue deleted on chromosome 10) gene, a tumour suppressor, were exposed to a 10% O2 (hypoxic) or 21% O2 (control) atmosphere for 7 days. Haematocrit, AST (aspartate aminotransferase), glucose, triacylglycerols (triglycerides) and insulin tolerance were measured in blood. Histological lesions were quantified. Expression of genes involved in lipogenesis and mitochondrial beta-oxidation, as well as FOXO1 (forkhead box O1), hepcidin and CYP2E1 (cytochrome P450 2E1), were analysed by quantitative PCR. In the animals exposed to hypoxia, the haematocrit increased (60+/-3% compared with 50+/-2% in controls; P<0.01) and the ratio of liver weight/body weight increased (5.4+/-0.2% compared with 4.7+/-0.3% in the controls; P<0.01). Furthermore, in animals exposed to hypoxia, steatosis was more pronounced (P<0.01), and the NAS [NAFLD (non-alcoholic fatty liver disease) activity score] (8.3+/-2.4 compared with 2.3+/-10.7 in controls; P<0.01), serum AST, triacylglycerols and glucose were higher. Insulin sensitivity decreased in mice exposed to hypoxia relative to controls. The expression of the lipogenic genes SREBP-1c (sterol-regulatory-element-binding protein-1c), PPAR-gamma (peroxisome-proliferator-activated receptor-gamma), ACC1 (acetyl-CoA carboxylase 1) and ACC2 (acetyl-CoA carboxylase 2) increased significantly in mice exposed to hypoxia, whereas mitochondria beta-oxidation genes [PPAR-alpha (peroxisome-proliferator-activated receptor-alpha) and CPT-1 (carnitine palmitoyltransferase-1)] decreased significantly. In conclusion, the findings of the present study demonstrate that hypoxia alone aggravates and accelerates the progression of NASH by up-regulating the expression of lipogenic genes, by down-regulating genes involved in lipid metabolism and by decreasing insulin sensitivity.

Targeting vessels to treat hepatocellular carcinoma.

The process of blood vessel proliferation, known as angiogenesis, is essential during embryonic development and organogenesis. In adult life, it participates in normal tissue repair, wound healing, and cyclical growth of the corpus luteum and the endometrium. Crucial as it is, angiogenesis can become pathological, and abnormal angiogenesis contributes to the pathogenesis of inflammatory and neoplasic diseases. The present review highlights the evidence for the role of angiogenesis in HCC (hepatocellular carcinoma) and discusses the increasing importance of inhibitors of angiogenesis in HCC therapy.

Effects on hepatocellular carcinoma of doxorubicin-loaded immunoliposomes designed to target the VEGFR-2.

To maintain a tumour vasculature in proportion of the tumour growth, the endothelial cells proliferate and up-regulate the expression of the VEGF receptor 2 (VEGFR-2), whose expression is restricted to this cell type. This specificity implies that one therapeutically target the tumour endothelium. We investigated the use of immunoliposomes (IL), containing conjugated Fab' fragments of the monoclonal rat anti-VEGFR-2 antibody DC101 (DC101-IL) to cargo doxorubicin to the tumour endothelium. In vitro, fluorescein-labelled IL displayed a 7 fold better binding to VEGFR-2-positive 293T cells in comparison to unspecific liposomes. Balb/C mice were injected subcutaneously with syngeneic hepatocellular carcinoma cells. One set of animals was treated with DC101-IL filled with doxorubicin when the tumours were bigger than 400 mm3. A specific delivery of doxorubicin to endothelial cells of the tumour vessels could be demonstrated by the red fluorescence of doxorubicin with laser scanning microscopy, but neither a delay of tumour growth nor a shrinking of the tumour mass was observed. Yet necrosis in the tumours treated with doxorubicin containing vehicles was larger than in the tumours of the control groups. A second set of animals was treated with DC101-IL filled with doxorubicin when the tumours were smaller than 1 mm3. DC101-IL filled with doxorubicin led to a significant delay in tumour growth up to 7 weeks compared to empty DC101-IL, free doxorubicin, and HEPES/Glucose (HEPES/Glucose vs. DOX-DC101-IL, p = 0.001; unpaired, two-tailed Student's t-test) and to a higher amount of necrotic areas in the tumours (p = 0.053; 1 way ANOVA with 4 groups). These findings suggest that IL designed to bind specifically to VEGFR-2 can be used to deliver doxorubicin to the tumour endothelium and may impair the "angiogenic switch" of the tumours.

Impact of MET targeting on tumor-associated angiogenesis and growth of MET mutations-driven models of liver cancer.

Deregulated expression of the MET receptor tyrosine kinase has been reported in up to 50% of patients with hepatocellular carcinoma, the most abundant form of liver cancers, and is associated with decreased survival. Consequently, MET is considered as a molecular target in this malignancy, whose progression is highly dependent on extensive angiogenesis. Here we studied the impact of MET small molecule inhibitors on angiogenesis-associated parameters and growth of xenograft liver models consisting of cells expressing MET-mutated variants M1268T and Y1248H, which exhibit constitutive kinase activity. We demonstrate that MET mutations expression is associated with significantly increased production of vascular endothelial growth factor, which is blocked by MET targeting only in cells expressing the M1268T inhibitor-sensitive but not in the Y1248H inhibitor-resistant variant. Decrease in vascular endothelial growth factor production is also associated with reduction of tyrosine phopshorylation of the vascular endothelial growth factor receptor 2 expressed on primary liver sinusoidal endothelial cells and with inhibition of vessel formation. Furthermore, MET inhibition demonstrated an efficient anti-tumor activity and considerable reduction in microvessel density only against the M1268T-derived intrahepatic tumors. Collectively, our data support the role of targeting MET-associated angiogenesis as a major biological determinant for liver tumor growth control.

TM6SF2 rs58542926 influences hepatic fibrosis progression in patients with non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is an increasingly common condition, strongly associated with the metabolic syndrome, that can lead to progressive hepatic fibrosis, cirrhosis and hepatic failure. Subtle inter-patient genetic variation and environmental factors combine to determine variation in disease progression. A common non-synonymous polymorphism in TM6SF2 (rs58542926 c.449 C>T, p.Glu167Lys) was recently associated with increased hepatic triglyceride content, but whether this variant promotes clinically relevant hepatic fibrosis is unknown. Here we confirm that TM6SF2 minor allele carriage is associated with NAFLD and is causally related to a previously reported chromosome 19 GWAS signal that was ascribed to the gene NCAN. Furthermore, using two histologically characterized cohorts encompassing steatosis, steatohepatitis, fibrosis and cirrhosis (combined n=1,074), we demonstrate a new association, independent of potential confounding factors (age, BMI, type 2 diabetes mellitus and PNPLA3 rs738409 genotype), with advanced hepatic fibrosis/cirrhosis. These findings establish new and important clinical relevance to TM6SF2 in NAFLD.

Study of the cellular mechanism of Sunitinib mediated inactivation of activated hepatic stellate cells and its implications in angiogenesis.

The development of hepatocellular carcinomas from malignant hepatocytes is frequently associated with intra- and peritumoral accumulation of connective tissue arising from activated hepatic stellate cells (HSC). Inhibition of receptor tyrosine kinase (RTK) signaling showed promise in the treatment of hepatocellular carcinoma. However, there is a lack of knowledge about the effects of RTK inhibitors on the tumor supportive cells. We performed in vitro experiments to study whether Sunitinib, a platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) RTKs' inhibitor, could block both activated HSC functions and angiogenesis and thus prevent the progression of cirrhotic liver to hepatocellular carcinoma. In immortalized human activated HSC LX-2, treatment with Sunitinib 100 nM blocked collagen synthesis by 47%, as assessed by Sirius Red staining, attenuated HSC contraction by 65%, and reduced cell migration by 28% as evaluated using a Boyden's chamber, without affecting cell viability, measured by Trypan blue staining, and apoptosis, measured by propidium iodide (PI) incorporation assay. Our data revealed that Sunitinib treatment blocked the transdifferentiation of primary human HSC (hHSC) to activated myofibroblast-like cells by 65% without affecting hHSC apoptosis and migration. In in vitro angiogenic assays, Sunitinib 100 nM reduced endothelial cells (EC) ring formation by 46% and tube formation by 68%, and decreased vascular sprouting in aorta ring assay and angiogenesis in vascular bed of chick embryo. In conclusion, the present study demonstrates that the RTK inhibitor Sunitinib blocks the activation of HSC and angiogenesis suggesting its potential as a drug candidate in pathological conditions like liver fibrosis and hepatocellular carcinoma.

Everolimus augments the effects of sorafenib in a syngeneic orthotopic model of hepatocellular carcinoma.

Sorafenib targets the Raf/mitogen-activated protein kinase, VEGF, and platelet-derived growth factor pathways and prolongs survival patients in advanced hepatocellular carcinoma (HCC). Everolimus inhibits the mammalian target of rapamycin, a kinase overactive in HCC. To investigate whether the antitumor effects of these agents are additive, we compared a combined and sequential treatment regimen of everolimus and sorafenib with monotherapy. After hepatic implantation of Morris Hepatoma (MH) cells, rats were randomly allocated to everolimus (5 mg/kg, 2×/week), sorafenib (7.5 mg/kg/d), combined everolimus and sorafenib, sequential sorafenib (2 weeks) then everolimus (3 weeks), or control groups. MRI quantified tumor volumes. Erk1/2, 4E-BP1, and their phosphorylated forms were quantified by immunoblotting. Angiogenesis was assessed in vitro by aortic ring and tube formation assays, and in vivo with Vegf-a mRNA and vascular casts. After 35 days, tumor volumes were reduced by 60%, 85%, and 55%, relative to controls, in everolimus, the combination, and sequential groups, respectively (P < 0.01). Survival was longest in the combination group (P < 0.001). Phosphorylation of 4E-BP1 and Erk1/2 decreased after everolimus and sorafenib, respectively. Angiogenesis decreased after all treatments (P < 0.05), although sorafenib increased Vegf-a mRNA in liver tumors. Vessel sprouting was abundant in control tumors, lower after sorafenib, and absent after the combination. Intussusceptive angiogenic transluminal pillars failed to coalesce after the combination. Combined treatment with everolimus and sorafenib exerts a stronger antitumoral effect on MH tumors than monotherapy. Everolimus retains antitumoral properties when administered sequentially after sorafenib. This supports the clinical use of everolimus in HCC, both in combination with sorafenib or after sorafenib.

Inhibition of mTOR in combination with doxorubicin in an experimental model of hepatocellular carcinoma.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is resistant to chemotherapy. We reported that sirolimus, an mTOR inhibitor, has antiangiogenic properties in HCC. Since antiangiogenic therapy may enhance chemotherapy effects, we tested the antitumorigenic properties of sirolimus combined with doxorubicin in experimental HCC. METHODS: Morris Hepatoma (MH) cells were implanted into livers of syngeneic rats. Animals were assigned to sirolimus, pegylated liposomal doxorubicin, both combined or control groups. Tumoral growth was followed by MRI. Antiangiogenic effects were assessed by CD31 immunostaining and capillary tube formation assays. Cell proliferation was monitored in vitro by thymidine incorporation. Expression of p21 and phosphorylated MAPKAP kinase-2 was quantified by immunoblotting. RESULTS: Animals treated with the combination developed smaller tumors with decreased tumor microvessel density compared to animals that received monotherapies. In vitro, inhibition of mTOR further impaired capillary formation in the presence of doxorubicin. Doxorubicin reduced endothelial cell proliferation; inhibition of mTOR accentuated this effect. Doxorubicin stimulated p21 expression and the phosphorylation of MAPKAP kinase-2 in endothelial cells. Addition of mTOR inhibitor down-regulated p21, but did not decrease MAPKAP kinase-2 phosphorylation. CONCLUSIONS: Sirolimus has additive antitumoral and antiangiogenic effects when administered with doxorubicin. These findings offer a rationale for combining mTOR inhibitors with chemotherapy in HCC treatment.

Vascular remodeling and antitumoral effects of mTOR inhibition in a rat model of hepatocellular carcinoma.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is amenable to only few treatments. Inhibitors of the kinase mTOR are a new class of immunosuppressors already in use after liver transplantation. Their antiproliferative and antiangiogenic properties suggest that these drugs could be considered to treat HCC. We investigated the antitumoral effects of mTOR inhibition in a HCC model. METHODS: Hepatoma cells were implanted into livers of syngeneic rats. Animals were treated with the mTOR inhibitor sirolimus for 4 weeks. Tumor growth was monitored by MR imaging. Antiangiogenic effects were assessed in vivo by microvessel density and corrosion casts and in vitro by cell proliferation, tube formation and aortic ring assays. RESULTS: Treated rats had significantly longer survival and developed smaller tumors, fewer extrahepatic metastases and less ascites than controls. Sirolimus decreased intratumoral microvessel density resulting in extensive necrosis. Endothelial cell proliferation was inhibited at lower drug concentrations than hepatoma cells. Tube formation and vascular sprouting of aortic rings were significantly impaired by mTOR inhibition. Casts revealed that in tumors treated with sirolimus vascular sprouting was absent, whereas intussusception was observed. CONCLUSIONS: mTOR inhibition significantly reduces HCC growth and improves survival primarily via antiangiogenic effects. Inhibitors of mTOR may have a role in HCC treatment.

Hint2, a mitochondrial apoptotic sensitizer down-regulated in hepatocellular carcinoma.

BACKGROUND & AIMS: Hints, histidine triad nucleotide-binding proteins, are adenosine monophosphate-lysine hydrolases of uncertain biological function. Here we report the characterization of human Hint2. METHODS: Tissue distribution was determined by real-time quantitative polymerase chain reaction and immunoblotting, cellular localization by immunocytochemistry, and transfection with green fluorescent protein constructs. Enzymatic activities for protein kinase C and adenosine phosphoramidase in the presence of Hint2 were measured. HepG2 cell lines with Hint2 overexpressed or knocked down were established. Apoptosis was assessed by immunoblotting for caspases and by flow cytometry. Tumor growth was measured in SCID mice. Expression in human tumors was investigated by microarrays. RESULTS: Hint2 was predominantly expressed in liver and pancreas. Hint2 was localized in mitochondria. Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L). Exposed to apoptotic stress, fewer HepG2 cells overexpressing Hint2 remained viable (32.2 +/- 0.6% vs 57.7 +/- 4.6%), and more cells displayed changes of the mitochondrial membrane potential (87.8 +/- 2.35 vs 49.7 +/- 1.6%) with more cleaved caspases than control cells. The opposite was observed in HepG2 cells with knockdown expression of Hint2. Subcutaneous injection of HepG2 cells overexpressing Hint2 in SCID mice resulted in smaller tumors (0.32 +/- 0.13 g vs 0.85 +/- 0.35 g). Microarray analyses revealed that HINT2 messenger RNA is downregulated in hepatocellular carcinomas (-0.42 +/- 0.58 log2 vs -0.11 +/- 0.28 log2). Low abundance of HINT2 messenger RNA was associated with poor survival. CONCLUSION: Hint2 defines a novel class of mitochondrial apoptotic sensitizers down-regulated in hepatocellular carcinoma.