hymecromone and Liver-Neoplasms

Reaching efficacious drug delivery to target cells/tissues represents a major obstacle in the current treatment of solid malignancies including hepatocellular carcinoma (HCC). In this study, we developed a pipeline to selective add complex-sugars to the aglycone 4-methylumbelliferone (4MU) to help their bioavailability and tumour cell intake.. The therapeutic efficacy of sugar-modified rutinosyl-4-methylumbelliferone (4MUR) and 4MU were compared in vitro and in an orthotopic HCC model established in fibrotic livers. The mechanistic bases of its selective target to liver tumour cells were evaluated by the interaction with asialoglycoprotein receptor (ASGPR), the mRNA expression of hyaluronan synthases (HAS2 or HAS3) and hyaluronan deposition.. 4MUR showed a significant antiproliferative effect on liver tumoural cells as compared to non-tumoural cells in a dose-dependent manner. Further analysis showed that 4MUR is incorporated mostly into HCC cells by interaction with ASGPR, a receptor commonly overexpressed in HCC cells. 4MUR-treatment decreased the levels of HAS2 and HAS3 and the cytoplasmic deposition of hyaluronan. Moreover, 4MUR reduced CFSC-2G activation, hence reducing the fibrosis. In vivo efficacy showed that 4MUR treatment displayed a greater tumour growth inhibition and increased survival in comparison to 4MU. 4MUR administration was associated with a significant reduction of liver fibrosis without any signs of tissue damage. Further, 60% of 4MUR treated mice did not present macroscopically tumour mass post-treatment.. Our results provide evidence that 4MUR may be used as an effective HCC therapy, without damaging non-tumoural cells or other organs, most probably due to the specific targeting.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Hyaluronan Synthases; Hymecromone; Liver Cirrhosis; Liver Neoplasms; Mice

Hepatocellular carcinoma (HCC) arises in the setting of advanced liver fibrosis, a dynamic and complex inflammatory disease. The tumor microenvironment (TME) is a mixture of cellular components including cancer cells, cancer stem cells (CSCs), tumor-associated macrophages (TAM), and dendritic cells (DCs), which might drive to tumor progression and resistance to therapies. In this work, we study the effects of 4-methylumbelliferone (4Mu) on TME and how this change could be exploited to promote a potent immune response against HCC. First, we observed that 4Mu therapy induced a switch of hepatic macrophages (Mϕ) towards an M1 type profile, and HCC cells (Hepa129 cells) exposed to conditioned medium (CM) derived from Mϕ treated with 4Mu showed reduced expression of several CSCs markers and aggressiveness. HCC cells incubated with CM derived from Mϕ treated with 4Mu grew in immunosuppressed mice while presented delayed tumor progression in immunocompetent mice. HCC cells treated with 4Mu were more susceptible to phagocytosis by DCs, and when DCs were pulsed with HCC cells previously treated with 4Mu displayed a potent antitumoral effect in therapeutic vaccination protocols. In conclusion, 4Mu has the ability to modulate TME into a less hostile milieu and to potentiate immunotherapeutic strategies against HCC.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Dendritic Cells; Disease Models, Animal; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Hymecromone; Immunity; Liver Cirrhosis; Liver Neoplasms; Mice; Neoplastic Stem Cells; Phagocytosis; Signal Transduction; Tumor Microenvironment; Tumor-Associated Macrophages; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) is a liver tumor that usually arises in patients with cirrhosis. Hepatic stellate cells are key players in the progression of HCC, as they create a fibrotic micro-environment and produce growth factors and cytokines that enhance tumor cell proliferation and migration. We assessed the role of endoplasmic reticulum (ER) stress in the cross-talk between stellate cells and HCC cells. Mice with a fibrotic HCC were treated with the IRE1α-inhibitor 4μ8C, which reduced tumor burden and collagen deposition. By co-culturing HCC-cells with stellate cells, we found that HCC-cells activate IREα in stellate cells, thereby contributing to their activation. Inhibiting IRE1α blocked stellate cell activation, which then decreased proliferation and migration of tumor cells in different in vitro 2D and 3D co-cultures. In addition, we also observed cell-line-specific direct effects of inhibiting IRE1α in tumor cells.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chemotaxis; Coculture Techniques; Endoribonucleases; Humans; Hymecromone; Liver Neoplasms; Liver Neoplasms, Experimental; Male; Mice; Protein Serine-Threonine Kinases; Tissue Scaffolds

The tumor microenvironment (TME) represents a complex interplay between different cellular components, including tumor cells and cancer stem cells (CSCs), with the associated stroma; such interaction promotes tumor immune escape and sustains tumor growth. Several experimental approaches for cancer therapy are focused on TME remodeling, resulting in increased antitumor effects. We previously demonstrated that the hyaluronan synthesis inhibitor 4-methylumbelliferone (4Mu) decreases liver fibrosis and induces antitumor activity in hepatocellular carcinoma (HCC). In this work, 4Mu, in combination with an adenovirus encoding interleukin-12 genes (AdIL-12), elicited a potent antitumor effect and significantly prolonged animal survival (p < 0.05) in an orthotopic HCC model established in fibrotic livers. In assessing the presence of CSCs, we found reduced mRNA levels of CD133

Topics: Animals; Antigen-Presenting Cells; Biomarkers; Carcinoma, Hepatocellular; CD47 Antigen; Cell Line, Tumor; Cytotoxicity, Immunologic; Disease Models, Animal; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Hymecromone; Interleukin-12; Liver Neoplasms; Lymphocytes, Tumor-Infiltrating; Mice; Neoplastic Stem Cells; Phagocytosis; T-Lymphocytes; Tumor Microenvironment; Xenograft Model Antitumor Assays

Cirrhosis is characterized by an excessive accumulation of extracellular matrix components including hyaluronic acid (HA) and is widely considered a preneoplastic condition for hepatocellular carcinoma (HCC). 4-Methylumbelliferone (4MU) is an inhibitor of HA synthesis and has anticancer activity in an orthotopic HCC model with underlying fibrosis. Our aim was to explore the effects of HA inhibition by 4MU orally administered on tumor microenvironment. Hepa129 tumor cells were inoculated orthotopically in C3H/HeJ male mice with fibrosis induced by thioacetamide. Mice were orally treated with 4MU. The effects of 4MU on angiogenesis were evaluated by immunostaining of CD31 and quantification of proangiogenic factors (vascular endothelial growth factor, VEGF, interleukin-6, IL-6 and C-X-C motif chemokine 12, CXCL12). IL-6 was also quantified in Hepa129 cells in vitro after treatment with 4MU. Migration of endothelial cells and tube formation were also analyzed. As a result, 4MU treatment decreases tumor growth and increased animal survival. Systemic levels of VEGF were significantly inhibited in 4MU-treated mice. Expression of CD31 was reduced after 4MU therapy in liver parenchyma in comparison with control group. In addition, mRNA expression and protein levels of IL-6 and VEGF were inhibited both in tumor tissue and in nontumoral liver parenchyma. Interestingly, IL-6 production was dramatically reduced in Kupffer cells isolated from 4MU-treated mice, and in Hepa129 cells in vitro. Besides, 4MU was able to inhibit endothelial cell migration and tube formation. In conclusion, 4MU has antitumor activity in vivo and its mechanisms of action involve an inhibition of angiogenesis and IL-6 production. 4MU is an orally available molecule with potential for HCC treatment.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Chemokine CXCL12; Endothelial Cells; Gene Expression Regulation, Neoplastic; Hymecromone; Interleukin-6; Kupffer Cells; Liver Cirrhosis; Liver Neoplasms; Male; Mice; Mice, Inbred C3H; Neovascularization, Pathologic; Platelet Endothelial Cell Adhesion Molecule-1; Signal Transduction; Survival Analysis; Thioacetamide; Vascular Endothelial Growth Factor A

We have previously demonstrated that a low dose of cyclophosphamide (Cy) combined with gene therapy of interleukin-12 (AdIL-12) has a synergistic, although limited, antitumoral effect in mice with colorectal carcinoma. The main mechanism involved in the efficacy of Cy+AdIL-12 was the induction of a specific immune response mediated by cytotoxic T lymphocytes. Our current aims were to evaluate the effects of 4-methylumbelliferone (4Mu), a selective inhibitor of hyaluronan (HA) synthesis, on tumor microenvironment (TME) and to investigate how 4Mu affects the therapeutic efficacy of Cy+AdIL-12. The results showed that 4Mu significantly reduced the amount of tumoral HA leading to a significant decrease in tumor interstitial pressure (TIP). As a consequence, tumor perfusion was improved allowing an increased adenoviral transgene expression. In addition, treatment with 4Mu boosted the number of cytotoxic T lymphocytes that reach the tumor after adoptive transfer resulting in a potent inhibition of tumor growth. Importantly, we observed complete tumor regression in 75% of mice when 4Mu was administrated in combination with Cy+AdIL-12. The triple combination 4Mu+Cy+AdIL-12 also induced a shift toward antiangiogenic factors production in tumor milieu. Our results showed that TME remodeling is an interesting strategy to increase the efficacy of anticancer immunotherapies based on gene and/or cell therapy.

Topics: Adenoviridae; Adoptive Transfer; Animals; Antineoplastic Agents, Alkylating; Cell Line, Tumor; Colorectal Neoplasms; Combined Modality Therapy; Cyclophosphamide; Cytotoxicity, Immunologic; Disease Models, Animal; Gene Expression; Genes, Reporter; Genetic Therapy; Genetic Vectors; Hymecromone; Immunotherapy; Interleukin-12; Liver Neoplasms; Lymphocytes, Tumor-Infiltrating; Male; Mice; Neovascularization, Pathologic; T-Lymphocyte Subsets; Transduction, Genetic; Transgenes; Tumor Burden; Tumor Microenvironment

Microenvironment around tumor cells plays an important role in its malignancy or invasiveness. Hyaluronan (HA), a major component of extracellular matrix is found to be elevated in most of cancerous niche/microenvironment and performs regulatory role in the progression of tumors and metastasis. Overexpression of the hyaladherin, hyaluronan-binding protein 1 (HABP1) in the hepatocarcinoma cells (HepG2) termed as HepR21 leads to enhanced cell proliferation with increased HA 'pool' associated with HA 'cables' indicating elevated tumorous potential under 2D culture conditions. For in vitro experimentation, scaffold based three dimensional niche modeling may have greater acceptance than conventional 2D culture condition. Thus, we have examined the influence of intrinsic properties of non-mulberry tropical tasar silk fibroin on the HepR21 cells in order to develop a 3D hepatocarcinoma construction to act as model. The scaffold of tasar silk fibroin of Antheraea mylitta when efficiently loaded with transformed hepatocarcinoma cells, HepR21; exhibits enhanced adhesiveness, viability, metabolic activity, proliferation and enlarged cellular morphology in 3D compared to its parent cell line HepG2, supporting the earlier observation made in 2D system. In addition, formation of multicellular aggregates, the indicator of tumor progression is also revealed in silk based 3D culture conditions. Further, the use of 4-MU (a hyaluronan synthase inhibitor) on HepR21 cells reduces the HA level and downregulates the expression of growth promoting factors like pAKT and PKC; while upregulating the expression of the tumor suppressor p53. Thus, 4-MU efficiently reduces the tumor potency associated with increased HA pool as well as HA cables and the effect of 4-MU doubling up as an anticancer agent in 2D and 3D are also comparable. The in vitro 3D multicellular model demonstrates the insight of hepatocarcinoma progression and offers the predictability of cellular response to transfection efficacy, drug treatment and therapeutic intervention.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Carrier Proteins; Drug Screening Assays, Antitumor; Fibroins; Hep G2 Cells; Humans; Hymecromone; Liver Neoplasms; Mitochondrial Proteins; Tissue Scaffolds; Up-Regulation

4-Methylumbelliferone (MU) inhibits the cell surface hyaluronan (HA) formation, and that such inhibition results in suppression of adhesion and locomotion of cultured melanoma cells. Here, we examine the effect of MU on melanoma cell metastasis in vivo. MU-treated melanoma cells showed both decreased cell surface HA formation and suppression of liver metastasis after injection into the mice. Oral administration of MU to mice decreased tissue HA content. These HA knock-down mice displayed suppressed liver metastasis. Thus, both cell surface HA of melanoma cells and recipient liver HA can promote liver metastasis, indicating that MU has potential as an anti-metastatic agent.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Cell Adhesion; Cell Movement; Female; Glucuronosyltransferase; Hyaluronan Synthases; Hymecromone; Liver Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Metastasis

The effects of synthesized 7-OH-4-CH(3)-coumarin on proliferation and differentiation of human hepatoma carcinoma cell line, SMMC-7721, were examined. Results showed that 7-OH-4-CH(3)-coumarin suppressed the proliferation of the SMMC-7721 cells in a dose-dependent manner, with an IC(50) value of 356 +/- 1.8 .M, while concentrations< or =200 mM could trigger differentiation. After treatment with this compound at 100 mM, the growth curve of human hepatoma cells decreased markedly. When treated with 50 and 100 mM, the cells' electrophoresis rate decreased from 2.2 mm/s/V/cm in the control group to 1.5 and 1.8 mm/s/V/cm, respectively, and the alpha-fetoprotein content decreased from 123 ng/mg in the control group to 68 and 45 ng/mg, respectively. The microvilli on the surface of treated cells were also reduced. All the above indexes related to cell malignancy were alleviated significantly. Results showed that 7-OH-4-CH(3)-coumarin could reverse human hepatoma cells' malignant phenotypic characteristics and induce redifferentiation.

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Cell Differentiation; Cell Division; Cell Line, Tumor; Cell Membrane; Cell Shape; Cell Survival; Coumarins; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Electrophoresis; Humans; Hymecromone; Inhibitory Concentration 50; Liver Neoplasms; Phenotype

A promising development in tumor therapy is the application of non-toxic prodrugs from which the active cytostatic is released by endogenous enzymes such as beta-glucuronidase (beta-gluc). Regulation of beta-gluc expression is one crucial factor modulating bioactivation of prodrugs. Recent experiments in rats indicate regulation of beta-gluc activity by the calcium channel blocker verapamil. To further explore this phenomenon, we investigated the effect of verapamil on beta-gluc enzyme activity, protein (western blot) and mRNA expression (RT-PCR) as well as the underlying mechanisms (effects of verapamil metabolites; promoter activity) in the human hepatoma cell line HepG2. Treatment of HepG2 cells with verapamil revealed down-regulation of beta-gluc activity, protein, and mRNA level down to 50% of the control with EC(50) values of 25 microM. Effects were similar for both enantiomers. Moreover, it was demonstrated that reduced promoter activity contributes to the observed effects. In summary, our data demonstrate regulation of human beta-glucuronidase expression by verapamil. Based on our findings we hypothesize that coadministration of verapamil may effect cleavage of glucuronides by beta-glucuronidase.

Topics: Blotting, Western; Calcium Channel Blockers; Carcinoma, Hepatocellular; Cell Line, Tumor; Down-Regulation; Genes, Reporter; Glucuronidase; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Hymecromone; Liver Neoplasms; Promoter Regions, Genetic; RNA, Messenger; Stereoisomerism; Time Factors; Transfection; Verapamil

(1) UDP-glucuronosyltransferase (UDP-GT) activities and their inducibility were investigated in human liver microsomes from a "liver bank". (2) UDP-GT activities were differentially induced in liver microsomes from patients treated with the phenobarbital-type inducers phenytoin or pentobarbital. UDP-GT activity towards bilirubin was induced 3-fold. Enzyme activities towards paracetamol, benzo(a)pyrene-3,6-quinol, 4-methylumbelliferone and 1-naphthol were moderately induced and to similar extents (2-fold). In contrast, morphine and 4-hydroxybiphenyl glucuronidation were not significantly affected. Cytochrome P-450 dependent 7-ethoxycoumarin O-deethylase was increased 5-fold. (3) A human hepatoma cell line (Hep G2) was studied to obtain information on the inducibility of human UDP-GT activities by 3-methylcholanthrene-type inducers. UDP-GT activities towards benzo(a)pyrene-3,6-quinol and 1-naphthol were moderately but significantly induced by 3-methylcholanthrene-treatment of the cells (2-fold), whereas 7-ethoxyresorufin O-deethylase and 7-ethoxycoumarin O-deethylase were increased over 100- and 10-fold, respectively. (4) The results suggest the existence of differentially inducible UDP-GT isoenzymes in human liver. The findings may be useful as a guide to characterize human liver UDP-GT isoenzymes.

Topics: Acetaminophen; Adolescent; Adult; Benzopyrenes; Bilirubin; Carcinoma, Hepatocellular; Enzyme Induction; Female; Glucuronosyltransferase; Humans; Hydroquinones; Hymecromone; Isoenzymes; Liver Neoplasms; Male; Methylcholanthrene; Microsomes, Liver; Middle Aged; Naphthols; Pentobarbital; Phenytoin; Tumor Cells, Cultured

Beta-Xylosides stimulate 2- to 6-fold the synthesis of glycosaminoglycans by three types of nonconnective tissue cells (RG-C6, NB41A, and rat hepatoma cells, and normal and simian virus 40 (SV40)-transformed normal human skin fibroblasts. The effect, which is specific for the anomeric linkage and the glycone, is observed in the presence and absence of puromycin. Beta-Xylosides may substitute for xylosylated core protein as initiators of synthesis of chondroitin sulfate chains. No stimulation of synthesis of heparan sulfate was observed. With the use of a fluorogenic xyloside, 4-methylumbelliferyl-beta-D-xyloside, it was demonstrated that the free chondroitin sulfate chains secreted into the medium bear the xyloside at the reducing end, and have an average molecular weight of 16,500.

Topics: Animals; Carcinoma, Hepatocellular; Cells, Cultured; Chondroitin; Chromatography, Gel; Dermatan Sulfate; Fibroblasts; Galactose; Glycosaminoglycans; Glycosides; Heparitin Sulfate; Humans; Hyaluronic Acid; Hymecromone; Liver Neoplasms; Mice; Neuroblastoma; Neuroglia; Nitrophenols; Rats; Simian virus 40; Skin; Spectrometry, Fluorescence; Sulfur Radioisotopes; Sulfuric Acids; Tritium; Xylose