M2 macrophages mediate sorafenib resistance by secreting HGF in a feed-forward manner in hepatocellular carcinoma.

BACKGROUND: Sorafenib is the only approved first line systemic therapy for advanced hepatocellular carcinoma (HCC) in the last decade. Tumour resistance to sorafenib has been of major obstacles to improve HCC patient survival. METHODS: We polarised THP-1 cells to M1 and M2 macrophages, performed various in vitro assays and developed sorafenib-resistant xenograft models to investigate the role of tumour-associated macrophages (TAM)-secreted molecules in HCC resistance to the targeted therapy. RESULTS: We demonstrated M2, but not M1, macrophages not only promote proliferation, colony formation and migration of hepatoma cells but also significantly confer tumour resistance to sorafenib via sustaining tumour growth and metastasis by secreting hepatocyte growth factor (HGF). HGF activates HGF/c-Met, ERK1/2/MAPK and PI3K/AKT pathways in tumour cells. Tumour-associated M2 macrophages were accumulated in sorafenib-resistance tumours more than in sorafenib-sensitive tumours in vivo and produced abundant HGF. HGF chemoattracts more macrophages migrated from surrounding area, regulates the distribution of M2 macrophages and increases hepatoma resistance to sorafenib in a feed-forward manner. CONCLUSIONS: Our results provide new insights into the mechanisms of sorafenib resistance in HCC and rationale for developing new trials by combining sorafenib with a potent HGF inhibitor such as cabozantinib to improve the first line systemic therapeutic efficacy.

Extracellular nucleotides as novel, underappreciated pro-metastatic factors that stimulate purinergic signaling in human lung cancer cells.

BACKGROUND: One of the challenging problems of current radio-chemotherapy is recurrence and metastasis of cancer cells that survive initial treatment. We propose that one of the unwanted effects of radiochemotherapy is the release from damaged ("leaky") cells of nucleotides such as ATP and UTP that exert pro-metastatic functions and can directly stimulate chemotaxis of cancer cells. METHODS: To address this problem in a model of human lung cancer (LC), we employed several complementary in vitro and in vivo approaches to demonstrate the role of extracellular nucleotides (EXNs) in LC cell line metastasis and tumor progression. We measured concentrations of EXNs in several organs before and after radiochemotherapy. The purinergic receptor agonists and antagonists, inhibiting all or selected subtypes of receptors, were employed in in vitro and in vivo pro-metastatic assays. RESULTS: We found that EXNs accumulate in several organs in response to radiochemotherapy, and RT-PCR analysis revealed that most of the P1 and P2 receptor subtypes are expressed in human LC cells. EXNs were found to induce chemotaxis and adhesion of LC cells, and an autocrine loop was identified that promotes the proliferation of LC cells. Most importantly, metastasis of these cells could be inhibited in immunodeficient mice in the presence of specific small molecule inhibitors of purinergic receptors. CONCLUSIONS: Based on this result, EXNs are novel pro-metastatic factors released particularly during radiochemotherapy, and inhibition of their pro-metastatic effects via purinergic signaling could become an important part of anti-metastatic treatment.

Low hepcidin triggers hepatic iron accumulation in patients with hepatitis C.

Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver disease including fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Chronic hepatitis C (CHC) is also a problem in patients with chronic kidney disease (CKD), particularly in those on haemodialysis. Excessive iron in the liver of CHC patients contributes to hepatic fibrosis, cirrhosis and finally HCC, while iron depletion is beneficial. In CHC patients without CKD, in HCV-infected experimental animals and in cell culture studies, serum hepcidin levels and/or cellular hepcidin expression are low and directly suppressed by HCV, radical oxygen species, growth factors and/or transcription factors. In contrast, antiviral therapy (e.g. with pegylated interferon-alpha combined with ribavirin) raises hepcidin levels and reduces iron overload in patients with CHC. Hepcidin directly inhibits HCV replication mediated by STAT3 activation. HCV circumvents hepatic innate antiviral defence by lowering hepcidin. If hepcidin is also low in CKD patients with CHC, iron supplementation should be avoided even in CKD patients with CHC treated with erythropoiesis-stimulating agents.

Herbacetin, a constituent of ephedrae herba, suppresses the HGF-induced motility of human breast cancer MDA-MB-231 cells by inhibiting c-Met and Akt phosphorylation.

Ephedrae herba suppresses hepatocyte growth factor-induced cancer cell motility by inhibiting tyrosine phosphorylation of the hepatocyte growth factor receptor, c-Met, and the PI3K/Akt pathway. Moreover, Ephedrae herba directly inhibits the tyrosine-kinase activity of c-Met. Ephedrine-type alkaloids, which are the active component of Ephedrae herba, do not affect hepatocyte growth factor-c-Met-Akt signalling, prompting us to study other active molecules in the herb. We recently discovered herbacetin glycosides and found that their aglycon, herbacetin, inhibits hepatocyte growth factor-c-Met-Akt signalling. This study revealed a novel biological activity of herbacetin. Herbacetin suppressed hepatocyte growth factor-induced motility in human breast cancer MDA-MB-231 cells by inhibiting c-Met and Akt phosphorylation and directly inhibiting c-Met tyrosine kinase activity. The effects of herbacetin were compared to those of kaempferol, apigenin, and isoscutellarein, all of which have similar structures. Herbacetin inhibition of hepatocyte growth factor-induced motility was the strongest of those for the tested flavonols, and only herbacetin inhibited the hepatocyte growth factor-induced phosphorylation of c-Met. These data suggest that herbacetin is a novel Met inhibitor with a potential utility in cancer therapeutics.

Hepatocyte growth factor has a role in the amelioration of diabetic vascular complications via autophagic clearance of advanced glycation end products: Dispo85E, an HGF inducer, as a potential botanical drug.

The aim of this study was to evaluate the effect and elucidate the potential mechanism of the extract of rhizomes from Dioscorea alata L. cv. Phyto, Dispo85E, on accelerating the elimination of advanced glycation end products (AGEs) in vitro and in vivo. Primary mouse nonparenchymal cells (NPCs) were used to evaluate the drug effect on AGEs clearance and autophagic-lysosomal activity. In an animal study, we used AGEs-induced diabetic mice to evaluate the drug effect on AGEs-induced vascular complications. Our results indicated that Dispo85E enhanced the endocytosis and degradation activity of AGEs in hepatic NPCs. Furthermore, the hepatocyte growth factor (HGF) expression level was positively correlated with the clearance capacity of the AGEs in NPCs after Dispo85E treatment. In addition, the effects of Dispo85E on the degradation and uptake capability of (14)C-AGEs were abolished in the presence of an anti-HGF neutralizing antibody. We further demonstrated that recombinant mouse HGF could enhance the endocytosis and autophagic clearance of AGEs in NPCs. The in vivo data indicated that Dispo85E increased hepatic HGF messenger RNA expression levels and decreased serum AGEs level in diabetic mice. Moreover, the function of retina and kidneys was improved by Dispo85E treatment in AGEs-induced diabetic mice. These results suggest that HGF may have an important role in the elimination of AGEs. This study suggests that Dispo85E is a botanical drug with a novel mechanism that enhances the clearance of AGEs through HGF-induced autophagic-lysosomal pathway and is a candidate drug for the treatment of diabetic vascular complications.

Effect of hepatocyte growth factor on endogenous hepatocarcinogenesis in rats fed a choline-deficient L-amino acid-defined diet.

Hepatocyte growth factor (HGF) is a promising agent for the treatment of intractable liver disease, due to its mitogenic, anti-apoptotic, and anti-fibrotic effects. We investigated the effect of recombinant human HGF (rh-HGF) on the development of both hepatocellular carcinoma (HCC) and preneoplastic nodules in rats fed a choline-deficient L-amino acid-defined (CDAA) diet, an animal model of hepatocarcinogenesis resembling human development of HCC with cirrhosis. From weeks 13 to 48 of the CDAA diet, rh-HGF (0.1 or 0.5 mg/kg/day) was administered intravenously to rats in four-week cycles, with treatment for five consecutive days of each week for two weeks, followed by a two-week washout period. Treatment with rh-HGF significantly inhibited the development of preneoplastic nodules in a dose-dependent manner at 24 weeks. Although the numbers and areas of the preneoplastic nodules in rats treated with rh-HGF were equivalent to those in mock-treated rats by 60 weeks, the incidence of HCC was reduced by HGF treatment. Although one rat treated with low-dose rh-HGF exhibited a massive HCC, which occupied almost the whole liver, and lung metastases, HGF treatment did not increase the overall frequency of HCC. Administration of high-dose rh-HGF, however, induced an increase in the urinary excretion of albumin, leading to decreased serum albumin at 60 weeks. These results indicate that long-term administration of rh-HGF does not accelerate hepatocarcinogenesis in rats fed a CDAA diet. However, these findings do not completely exclude the potential of HGF-induced hepatocarcinogenesis; this issue must be resolved before rh-HGF can be used for patients with intractable liver diseases, especially those with cirrhosis.

Inhibition by evodiamine of hepatocyte growth factor-induced invasion and migration of tumor cells.

Tumor cell motility plays a crucial role in the establishment of tumor metastasis and is affected by a variety of host-derived factors during the event. Hepatocyte growth factor (HGF) is one of these factors and stimulates tumor cell migration remarkably. We previously reported that evodiamine has a marked inhibitory activity on tumor cell invasion and migration in vitro. In this study, the effects of evodiamine on HGF-induced invasion and migration of tumor cell lines, colon 26-L5 carcinoma, B16-F10 melanoma and Lewis lung carcinoma (LLC) were examined. HGF promoted invasive activity of tumor cell lines with maximal induction of 1.8 times at 30 ng/ml for colon 26-L5 and LLC cells, and 2.0 times at 10 ng/ml for B16-F10 cells. Evodiamine inhibited the HGF-stimulated tumor cell invasion and migration in a concentration-dependent manner, and achieved complete suppression at 30 microM in all of the cell lines tested. When tumor cells were seeded on fibronectin-coated plates with evodiamine, their spreading on the plate was obviously inhibited, while their adhesiveness to fibronectin was unaffected. Evodiamine showed a marginal effect on tumor cell growth in a 24-h incubation, although it exhibited a marked inhibition in an over 48-h incubation. These results suggest that evodiamine suppressed HGF-stimulated invasion and migration of tumor cells partly through inhibition of cell spreading.

Significance of branched chain amino acids as possible stimulators of hepatocyte growth factor.

Amino acids can serve as regulatory molecules that modulate numerous cellular functions. Branched chain amino acids (BCAAs) are known to exert influences on cellular metabolism, amino acid transport, protein turn over, and gene expression. However, the mechanisms involved in the specific effect of BCAAs have not been clarified. BCAA supplementation therapy is a current treatment for patients with liver cirrhosis, therefore, specific BCAA activities should be examined. Hepatocyte growth factor (HGF) is considered to be a pleiotropic factor, and is reported to modulate gene expression and to stimulate the proliferation and functions of many cell types, including hepatocytes. A potential application of HGF for several types of diseases has been postulated. Here, we describe the potential of BCAAs as a therapeutic agent that acts through the induction of HGF production in the liver.

Nonsteroidal anti-inflammatory drugs for treatment of advanced gastric cancer: cyclooxygenase-2 is involved in hepatocyte growth factor mediated tumor development and progression.

Surgical treatment of gastric cancer patients is dismal because advanced tumor is often noted at diagnosis. In order to obtain better adjuvant therapy for gastric cancer patients after operation, it is important to understand the mechanism of invasion and metastasis. It is well known that binding of hepatocyte growth factor (HGF) to its receptor (c-Met) regulates gastric cancer progression and metastasis. Recently, HGF was found to up-regulate the expression of cyclooxygenase-2 (COX-2) gene and increase prostaglandin (PG)synthesis in gastric mucosa cells. Over-expression of COX-2 and increased PG secretion have also been found to be involved in the growth and metastasis of gastric cancer. These results together suggest that the signaling pathway of HGF and c-Met may be mediated through ERK2 activation, up-regulation of COX-2 and increased production of PGE(2)in gastric cancer cells. In view of the fact that c-Met is over-expressed in the majority of gastric cancer patients with poor prognosis, COX-2 specific inhibitors may provide beneficial effects in these patients.

Characterization of the functional domains of galactosylceramide expression factor 1 in MDCK cells.

We previously reported that GalCer expression factor 1 (GEF-1), a rat homologue of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), induced GalCer expression, morphological changes, and cell growth inhibition in COS-7 cells. In this study, we describe the characterization of GEF-1 in MDCK cells. Overexpression of GEF-1 in MDCK (MDCK/GEF-1) cells showed GalCer-derived sulfatide expression as well as dramatic morphological changes, but not cell growth suppression. The enzyme activity and the mRNA level of UDP-galactose:ceramide galactosyltransferase (CGT) increased significantly in MDCK/GEF-1 cells compared with control cells. GEF-1 molecule is composed of four domains; a zinc-finger (Z), a proline-rich (P), a coiled-coil (C), and a proline/glutamine-rich (Q) domain. MDCK cells transfected with various GEF-1 deletion mutants were examined for morphology and for glycolipid expression. MDCK cells transfected with Z-domain deletion mutant (MDCK/PCQ) and those with both Z- and P-domains deletion mutant (MDCK/CQ) were similar to those with a wild-type GEF-1 (MDCK/ZPCQ) in shape, exhibiting fibroblast-like cells, whereas those with the other deletion mutants showed no morphological changes, exhibiting typical epithelial-like cells. On the other hand, MDCK/ZPCQ, MDCK/PCQ, MDCK/CQ, and MDCK/Q cells expressed sulfatide, whereas those with the other deletion mutants that did not include the Q-domain showed neither GalCer nor sulfatide expression. Thus, the correlation between fibroblast-like cells in shape and the glycolipid expression was good in these deletion mutants except MDCK/Q cells, which showed epithelial-like cells, but expressed sulfatide. The glycolipid expression paralleled CGT mRNA levels. Taking these results together, it is suggested that only the Q-domain may be essential for the role of GEF-1 in inducing CGT mRNA, whereas the Q-domain together with the C-domain may be required for the induction of morphological changes in MDCK cells.

Expression of the met receptor tyrosine kinase in muscle progenitor cells in somites and limbs is absent in Splotch mice.

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates proliferation, dissociation, migration and morphogenesis of cells in culture. To investigate a possible role for HGF/SF and its receptor, the Met tyrosine kinase, in embryonic development, we have analyzed their expression in mouse embryos from day 7.5 of gestation by whole-mount in situ hybridization. Met expression is first detected in the ventral portion of somites at day 9.25 of gestation (22 somite embryo) at the level of fore limb buds. As somites mature, met expression is detected in caudal somites, and is confined to the lateral and media] tips of the dermomyotome and dermomyotome/myotome respectively. In contrast, HGF/SF is expressed exclusively in the mesodermal core of the limb bud. As the dermomyotome elongates ventrolaterally, the met-expressing cells at the lateral tip appear to detach from the somite, invade the limb bud and localize at the dorsal and ventral limb sides in close proximity to HGF/SF-expressing cells. At later stages, both met- and HGF/SF-expressing cells appear to migrate distally and localize to the digit forming area of the developing hand plate. Met expression in the lateral dermomyotome and limb bud coincides with expression of Pax-3, a marker for migrating muscle precursor cells in the somite and limb. Splotch-2H and Splotch-delayed mice, which harbor mutations in Pax-3, show major disruptions in early limb muscle development. Significantly, no met-expressing cells were observed in the limbs of homozygous Splotch-2H and Splotch-delayed animals, whereas HGF/SF expression was not affected. The restricted expression of met to a sub-population of Pax-3-expressing cells in the lateral tip of the dermomyotome, demonstrates that met represents a unique molecular marker for this migratory cell population. From these observations, together with the biological activities of HGF/SF, we propose that in homozygous Splotch embryos the failure of muscle precursors to migrate into and populate the limb bud results from a loss of met expression in the cells at the ventrolateral edge of the somitic dermomyotome.