Plerixafor (AMD3100) induces prolonged mobilization of acute lymphoblastic leukemia cells and increases the proportion of cycling cells in the blood in mice.

The CXCR4 antagonist Plerixafor (AMD3100) induces the rapid mobilization of hematopoietic stem and progenitor cells into the blood in mice and humans. AMD3100 similarly induces the mobilization of human acute lymphoblastic leukemia (ALL) cells into the blood in mice. In this study, the temporal response of pre-B ALL cells to AMD3100 was compared with that of normal hematopoietic progenitor cells (HPC) using an NOD/SCID xenograft model of ALL and BALB/c mice, respectively. ALL cells remained in the circulation up to 6 hours after AMD3100 administration, by which time normal HPCs were no longer detectable. AMD3100 also increased the proportion of actively cycling ALL cells in the peripheral blood. Together, these data suggest that ALL cells are more sensitive to the effects of bone marrow disruption than normal progenitors. Using the NOD/SCID xenograft model, we demonstrated that AMD3100 increased the efficacy of the cell cycle specific drug vincristine, resulting in reduced disease levels in the blood and spleens of animals over 3 weeks and extended the survival of NOD/SCID mice with ALL. These data demonstrate that mobilizing agents can increase the therapeutic effect of cell cycle dependent chemotherapeutic agents.

CXCR4 mediates the homing of B cell progenitor acute lymphoblastic leukaemia cells to the bone marrow via activation of p38MAPK.

The mechanisms regulating the migration of leukaemic cells between the blood and bone marrow compartments remain obscure, but are of fundamental importance for the dissemination of the disease. This study investigated the in vivo homing of human B cell progenitor acute lymphoblastic leukaemia (ALL) cells to the femoral bone marrow of non-obese diabetic severe combined immunodeficient (NOD/SCID) mice. It was demonstrated that patient ALL cells use the chemokine axis, chemokine (CXC motif) receptor 4 (CXCR4)/ chemokine (CXC motif) ligand 12 (CXCL12), to home to the femoral marrow. CXCL12-mediated signalling through p38 mitogen-activated protein kinase (MAPK) was required for optimal homing. In contrast, the homing of normal peripheral blood CD34(+) cells and the cytokine-dependent CD34(+) cell line Mo7e was independent of p38MAPK, consistent with the dependence of these cells, as well as normal CD34(+) CD19(+) B cell progenitors, on PI-3K/AKT signalling. Altogether, our data provide clarification of the direct role of CXCL12 in the bone marrow homing of ALL cells and demonstrate unique signalling molecule usage that may have therapeutic implications for this disease.

Apoptosis in experimental NASH is associated with p53 activation and TRAIL receptor expression.

BACKGROUND AND AIMS: We examined extrinsic and intrinsic (endogenous) mitochondrial apoptosis pathways in experimental non-alcoholic steatohepatitis (NASH). METHODS: To assess extrinsic pathways, we measured hepatic expression of death-inducing cytokine receptors (tumor necrosis factor-alpha-receptor (TNF-R)1, TNF-R2, Fas, and TNFalpha-related apoptosis-inducing ligand-receptor (TRAIL-R) mRNA, TUNEL, caspase 3 activation, liver injury and liver pathology in mice fed a methionine and choline deficient (MCD) diet. For endogenous stress pathways, we determined serum insulin-like growth factor-1 (IGF-1), hepatic p53, Bcl-XL, tBid and p21 expression. RESULTS: Methionine and choline deficient feeding increased alanine aminotransferase (ALT) and apoptosis from day 10, without increases in TNF-R1, TNF-R2, and Fas. However, murine TRAIL receptors, particularly decoyTRAIL-R1/TNFRSFH23 and Killer/DR5 mRNA increased. MCD feeding enhanced hepatic p53 expression, corresponding to approximately 50% fall in serum IGF-1, decreased Bcl-XL, enhanced Bid cleavage to tBid, and up-regulation of p21. Nutritional restitution experiments showed that correcting either methionine or choline deficiency suppressed liver inflammation (extrinsic pathway), but failed to correct apoptosis, IGF-1 or p53. CONCLUSIONS: Methionine and choline deficiency lower IGF-1 to de-repress p53 during induction of steatohepatitis. The p53 induced by nutritional stress is biologically active in mediating mitochondrial cell death pathways, but may also be responsible for TRAIL receptor expression, thereby linking intrinsic and exogenous apoptosis pathways in NASH.

Activation of peroxisome proliferator-activated receptor alpha by dietary fish oil attenuates steatosis, but does not prevent experimental steatohepatitis because of hepatic lipoperoxide accumulation.

BACKGROUND AND AIM: Non-alcoholic fatty liver disease is the result of an imbalance in hepatic lipid partitioning that favors fatty acid synthesis and storage over fatty acid oxidation and triglyceride secretion. The progressive, inflammatory disorder of steatohepatitis can be prevented or reversed by correcting this lipid imbalance by activating peroxisome proliferator-activated receptor (PPAR) alpha, a transcription factor which regulates fatty acid oxidation. n-3 polyunsaturated fatty acids (PUFA), such as those found in fish oil (FO), are naturally occurring PPARalpha ligands which also suppress lipid synthesis. METHODS: We tested the role of dietary activation of PPARalpha by feeding mice a n-3 PUFA-enriched FO diet in the methionine and choline deficient (MCD) model of steatohepatitis. Results were compared with mice fed the corresponding diet supplemented with monounsaturated fatty acids as olive oil (OO). RESULTS: As expected, FO feeding led to robust hepatic PPARalpha activation in control mice, and decreased expression of genes involved with fatty acid synthesis. Such lipolytic gene expression profile was also clearly evident in FO MCD-fed mice, and was associated with reduced hepatic lipid accumulation in comparison with mice fed OO MCD diet. FO feeding in control mice also caused marked hepatic accumulation of lipoperoxides compared with OO and chow-fed mice. This was further exacerbated in FO MCD-fed animals, which developed steatohepatitis characterized by mild steatosis and moderate inflammation in comparison with OO MCD-fed mice; such inflammatory recruitment was not related to NF-kappaB activation or enhanced cyclooxygenase-2 activity. CONCLUSIONS: Feeding an n-3 PUFA-enriched diet activated PPARalpha and suppressed hepatic de novo lipogenesis, but failed to prevent development of steatohepatitis in the presence of methionine and choline deficiency. Instead, the very high levels of hepatic lipoperoxides may have abrogated the protection that would otherwise be conferred by PPARalpha activation, and could also be responsible for lipotoxic hepatocellular injury and inflammatory recruitment.

NADPH oxidase is not an essential mediator of oxidative stress or liver injury in murine MCD diet-induced steatohepatitis.

BACKGROUND/AIMS: Hepatic oxidative stress is a key feature of metabolic forms of steatohepatitis, but the sources of pro-oxidants are unclear. The NADPH oxidase complex is critical for ROS generation in inflammatory cells; loss of any one component (e.g., gp91phox) renders NADPH oxidase inactive. We tested whether activated inflammatory cells contribute to oxidant stress in steatohepatitis. METHODS: gp91phox-/- and wildtype (wt) mice were fed a methionine and choline-deficient (MCD) diet. Serum ALT, hepatic triglycerides, histopathology, lipid peroxidation, activation of NF-kappaB, expression of NF-kappaB-regulated genes and macrophage chemokines were measured. RESULTS: After 10 days of MCD dietary feeding, gp91phox-/- and wt mice displayed equivalent hepatocellular injury. After 8 weeks, there were fewer activated macrophages in livers of gp91phox-/- mice than controls, despite similar mRNA levels for MCP and MIP chemokines, but fibrosis was similar. NF-kappaB activation and increased expression of ICAM-1, TNF-alpha and COX-2 mRNA were evident in both genotypes, but in gp91phox-/- mice, expression of these genes was confined to hepatocytes. CONCLUSIONS: A functional NADPH oxidase complex does not contribute importantly to oxidative stress in this model and therefore is not obligatory for induction or perpetuation of dietary steatohepatitis.

COX-2 induction in mice with experimental nutritional steatohepatitis: Role as pro-inflammatory mediator.

The underlying mechanisms that perpetuate liver inflammation in nonalcoholic steatohepatitis are poorly understood. We explored the hypothesis that cyclooxygenase-2 (COX-2) can exert pro-inflammatory effects in metabolic forms of fatty liver disease. Male wild-type (WT) C57BL6/N or peroxisome proliferator-activated receptor alpha knockout (PPAR-alpha-/-) mice were fed a lipogenic, methionine- and choline-deficient (MCD) diet or the same diet with supplementary methionine and choline (control). COX-2 was not expressed in livers of mice fed the control diet. In mice fed the MCD diet, hepatic expression of COX-2 messenger RNA and protein occurred from day 5, continued to rise, and was 10-fold higher than controls after 5 weeks, thereby paralleling the development of steatohepatitis. Upregulation of COX-2 was even more pronounced in PPAR-alpha-/- mice. Induction of COX-2 was completely prevented by dietary supplementation with the potent PPAR-alpha agonist Wy-14,643 in WT but not PPAR-alpha-/- mice. COX-2 upregulation was preceded by activation of nuclear factor kappaB (NF-kappaB) and coincided with increased levels of tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-6, and intercellular adhesion molecule 1 (ICAM-1). Selective COX-2 inhibitors (celecoxib and NS-398) protected against the development of steatohepatitis in WT but not PPAR-alpha-/- mice. In conclusion, induction of COX-2 occurs in association with NF-kappaB activation and upregulation of TNF-alpha, IL-6, and ICAM-1 in MCD diet-induced steatohepatitis. PPAR-alpha suppresses both COX-2 and development of steatohepatitis, while pharmacological inhibition of COX-2 activity ameliorates the severity of experimental steatohepatitis. COX-2 may therefore be a pro-inflammatory mediator in metabolic forms of steatohepatitis.

NF-kappaB activation, rather than TNF, mediates hepatic inflammation in a murine dietary model of steatohepatitis.

BACKGROUND & AIMS: We explored the roles of nuclear factor-kappaB (NF-kappaB) and tumor necrosis factor (TNF) alpha (TNF-alpha) as mediators of inflammation in a nutritional model of steatohepatitis. METHODS: Wild-type (wt), TNF null -/-, and TNF receptor (R)-1-/- mice were fed a methionine- and choline-deficient (MCD) diet for up to 5 weeks. Liver injury (serum alanine aminotransferase [ALT]), hepatic inflammation, triglycerides, and lipid peroxide levels were determined. Hepatic NF-kappaB activation and expression of TNF and intercellular adhesion molecule-1 (ICAM-1) were assayed. RESULTS: Irrespective of genotype, MCD diet-fed mice developed hepatic lipid peroxidation and serum ALT elevation; at day 10, livers from wt, TNF-/-, and TNFR-1-/- mice showed equivalent steatohepatitis. NF-kappaB/DNA binding was enhanced in hepatic nuclear fractions from MCD diet-fed wt mice compared with dietary controls; there were corresponding increases of ICAM-1 and TNF messenger RNA (mRNA). Likewise, NF-kappaB activation and ICAM-1 expression were enhanced by MCD dietary feeding in TNF-/- and TNFR-1-/- mice compared with respective controls. To establish whether NF-kappaB is a primary mediator of inflammation in experimental steatohepatitis, we over-expressed a mutant, nondegradable IkappaB (mIkappaB), delivered by adenovirus in vivo. As expected, hepatic mIkappaB expression reduced NF-kappaB/DNA binding induced by MCD dietary feeding, with resultant abrogation of ICAM-1 and TNF synthesis. Such blockade of NF-kappaB transcriptional activation substantially protected against development of steatohepatitis, with significant reductions in liver injury and hepatic inflammation. CONCLUSIONS: In the MCD dietary model of steatohepatitis, NF-kappaB is activated early and is an important proinflammatory mediator of lesion development, but steatohepatitis occurs independently of TNF synthesis and TNFR-1 activation.

Curcumin inhibits NF-kappaB activation and reduces the severity of experimental steatohepatitis in mice.

BACKGROUND/AIMS: While oxidative stress is a feature of non-alcoholic steatohepatitis, the causal link between oxidative stress and inflammatory recruitment has yet to be demonstrated. We analysed the role of NF-kappaB redox-sensitive signalling pathway of inflammatory recruitment in experimental steatohepatitis. METHODS: Mice were fed the methionine and choline deficient (MCD) or the control diet, with or without curcumin, an NF-kappaB inhibitor, for up to 4 weeks. Histopathology, lipoperoxides, NF-kappaB/DNA binding and expression of NF-kappaB-regulated genes were assessed. RESULTS: MCD-fed mice developed steatohepatitis accompanied by dramatic accumulation of hepatic lipoperoxides, activation of NF-kappaB and induction of pro-inflammatory ICAM-1, COX-2, MCP-1 and CINC mRNA. Curcumin significantly reduced MCD-induced inflammation but had no effect on steatosis or on the level of hepatic lipid peroxides. Curcumin prevented the MCD-induced activation of NF-kappaB and decreased downstream induction of ICAM-1, COX-2 and MCP-1. However, it failed to reduce activation of AP-1, MAPK pathways or CINC expression. CONCLUSIONS: Curcumin alleviates the severity of hepatic inflammation in experimental steatohepatitis induced by the MCD diet, an effect likely to be mediated via inhibition of NF-kB activation and dependent pro-inflammatory genes. The NF-kappaB pathway is one among several possible signalling pathways by which inflammation is recruited in experimental steatohepatitis.

Hepatic ischemic preconditioning in mice is associated with activation of NF-kappaB, p38 kinase, and cell cycle entry.

A brief period of hepatic ischemia protects the liver against subsequent ischemia-reperfusion (IR) injury, but the mechanism of such preconditioning is poorly understood. We examined whether preconditioning activated nuclear factor kappa B (NF-kappaB), the stress-activated protein kinases (SAPK), c-Jun N-terminal kinase-1 (JNK-1) and p38, and entry into the cell cycle. We used a murine model of partial hepatic ischemia. Preconditioning was performed by clamping the vasculature for 2 to 20 minutes, and allowing reperfusion for 10 minutes before 90-minute ischemia or IR. As assessed by serum alanine aminotransferase (ALT) levels and liver histology, preconditioning periods of 5 and 10 minutes were highly protective against IR injury, whereas 2-, 15-, and 20-minute intervals were ineffective. Preconditioning was associated with entry of hepatocytes into the cell cycle within 2 hours of subsequent IR, as indicated by proliferating cell nuclear antigen (PCNA) nuclear staining, induction of cyclin D1 and numerous mitotic figures; in the absence of preconditioning, such changes were not seen until 24 hours. Preconditioning increased nuclear binding of NF-kappaB within 30 minutes of the subsequent ischemic interval, paralleled by degradation of inhibitory (binding) protein for NF-kappaB (IkappaBalpha). Ischemic preconditioning also activated p38 kinase and JNK-1, which are known to converge on cyclin D1 regulation. The protective effect of the preconditioning regimen was more closely associated with p38 kinase than JNK-1 activation. In conclusion, the hepatoprotective effects of ischemic preconditioning are associated with activation of NF-kappaB and SAPKs that are associated with entry of hepatocytes into the cell cycle, a critical biological effect that favors survival of the liver against ischemic and IR injury.