manumycin and Carcinoma--Hepatocellular

Farnesyl-transferase inhibitor manumycin has in vitro and in vivo antitumor effects on pancreatic cancer, colon cancer, and anaplastic thyroid carcinoma. Our previous experiments showed that manumycin could inhibit proliferation pathway and survival pathway in human hepatocellular carcinoma HepG2 cells in vitro. This study was to examine the antitumor and anti-angiogenic effects of manumycin on HepG2 xenografts in nude mice.. The xenografts derived from HepG2 cells were established in BALB/C nude mice. Inoculated mice were randomly divided into normal saline (NS) group, positive control (cyclophosphamide, CTX, 25 mg/kg) group, negative control (0.1% Me2SO, 20 ml/kg) group, low dose munumycin (2.5 mg/kg) group, and high dose munumycin (5 mg/kg) group. Tumor volume was measured in nude mice bearing xenografts. Microvessel density (MVD) was observed by immunohistochemistry. Protein levels of vascular endothelial growth factor (VEGF) and b-fibroblast growth factor (b-FGF) were determined by Western blot; mRNA level of VEGF was analyzed using reverse transcription-polymerase chain reaction (RT-PCR).. The mean tumor volume ratio of nude mice xenograft (V/V(0)) was significantly lower in low dose manumycin group and high dose manumycin group than in negative control group (0.68+/-0.09 and 0.59+/-0.04 vs. 1.38+/-0.21, P < 0.01). MVD was significantly lower in manumycin-treated groups than in control group (P < 0.01). Manumycin significantly down-regulated protein level of VEGF in HepG2 cells and HepG2 xenografts, and mRNA level of VEGF in HepG2 xenografts, but didn't affect protein level of b-FGF.. Manumycin could inhibit the growth of human hepatocellular carcinoma HepG2 xenografts in nude mice. The down-regulation of VEGF expression and the inhibition of angiogenesis might play a key role in the anti-neoplastic effect of manumycin.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Dose-Response Relationship, Drug; Female; Fibroblast Growth Factor 2; Humans; Liver Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Microcirculation; Neoplasm Transplantation; Neovascularization, Pathologic; Polyenes; Polyunsaturated Alkamides; Random Allocation; RNA, Messenger; Vascular Endothelial Growth Factor A

Manumycin was reported to have inhibitory effect on farnesyltransferase by competing with the farnesyl pyrophosphate substrate. It exhibited different antiproliferative activity in human hepatocellular carcinoma HepG2 cells, primary cultured human cardiac muscle cells and human liver cells (CLC). HepG2 cells overexpressing ras gene were more sensitive to manumycin than the other cells. The difference might be related to Ras protein levels in these cell lines. Manumycin reduced the amount of functional ras localized at the cytoplasmic membrane, resulting in blocked C-raf-1 assocation with Ras. Manumycin inhibited ERK1/2 phosphorylation in HepG2 cells without reduced expression of ERK1/2 protein. The levels of protein MKP-1 were significantly up-regulated. Our study also demonstrated that manumycin inhibited p85/PI3K and Akt phosphorylation without reduced expression of p85/PI3K and Akt, and interfered with the association of p85/PI3K and Ras. These findings indicated that manumycin interfered with Ras membrane localization, shut down the downstream pathways of Ras and inhibited cell proliferation in HepG2 cells.

Topics: Alkyl and Aryl Transferases; Carcinoma, Hepatocellular; Cell Cycle Proteins; Cell Division; Cell Line; Cell Line, Tumor; Dual Specificity Phosphatase 1; Enzyme Inhibitors; Farnesyltranstransferase; Hepatocytes; Humans; Immediate-Early Proteins; Liver Neoplasms; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Myocytes, Cardiac; Phosphoinositide-3 Kinase Inhibitors; Phosphoprotein Phosphatases; Polyenes; Polyunsaturated Alkamides; Protein Phosphatase 1; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-raf; ras Proteins; Signal Transduction

Farnesyltransferase inhibitors (FTIs) were developed to prevent Ras processing and thus to be effective agents for the treatment of cancers harbouring mutated ras. In the present study, HepG2 cells underwent internucleosomal DNA fragmentation after treatment with farnesyltransferase inhibitor manumycin (20 microM) for 12 h. Flow cytometric analysis showed that HepG2 cells were accumulated in the G2/M phase of the cell cycle and the number of apoptotic sub-G1 fraction of cells was increased after treatment with manumycin in a time-dependent manner. During the induction of apoptosis, expression of p53 and p21WAF1 was upregulated, phosphorylation of IkappaB-alpha was blocked, caspase substrates poly(ADP-ribose) polymerase (PARP) and lamin B were cleaved, and Bcl-2 and Bax protein expression remained unchanged. These results indicated that manumycin induced apoptosis in HepG2 cells. The induction of apoptosis by manumycin involved the upregulation of p53 and p21WAF1, the activation of caspases, and the inhibition of nuclear factor-kappaB (NF-kappaB) pathway. However, Bcl-2 and Bax are not associated with manumycin-mediated apoptosis.

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Cycle; DNA Damage; Enzyme Inhibitors; Flow Cytometry; Humans; NF-kappa B; Polyenes; Polyunsaturated Alkamides; ras Proteins

Treatment with anti-cancer agents has failed to achieve satisfactory results in hepatocellular carcinoma. In the process of hepatocarcinogenesis, Ras has been shown to play an important role. Ras requires a farnesyl moiety for activation. It has been found that farnesyltransferase inhibitor Manumycin inhibits farnesyl protein transferase, which catalyzes farnesylation. This study was designed to investigate the antitumor effect of Manumycin in human hepatoma cell line HepG2 and try to clarify its influence on Ras pathway.. The growth inhibitory effect of farnesyltransferase inhibitor Manumycin on human hepatoma cell line HepG2 was observed by using [3H]thymidine incorporation assay. The relative protein expressions of pan-Ras, N-Ras, ERK1/2, AKT, and MKP-1 affected by Manumycin were determined by using Western blot analysis.. Manumycin(5, 10, 20, 40, and 80 mumol/L) significantly inhibited cell growth of human hepatoma cell line HepG2 with IC50 value of (17.1 +/- 2.6) mumol/L. Manumycin could inhibit both pan-Ras and N-Ras in human hepatoma HepG2 cells, but its inhibitory effect on pan-Ras of cell membrane was much stronger. Phospho-MAPK and phospho-AKT decreased significantly after treatment of HepG2 cells with Manumycin, while total MAPK and AKT were hardly affected. After treatment with 10 nmol/L wortmannin for 1 h, which had potent inhibitory effect on phosphorylation of AKT, Manumycin had stronger inhibitory effect on phosphorylation of AKT as compared to treatment without wortmannin, eventhough AKT protein levels were still unaffected. Furthermore, the expression of MKP-1 was elevated through manumycin treatment in a concentration-dependent manner.. Manumycin may significantly inhibit the growth of human hepatoma cell line HepG2, which was related to its inhibition on the combination of Ras and cell membrane and increasing the expression of MKP-1, accordingly inhibiting activation of ERK1/2 and AKT. These results suggest that Manumycin antagonizes the growth of HepG2 via the suppression of ras farnesylation blocking the function of oncogenic ras against and could be a potential new anti-cancer agents human cancer, including hepatocellular carcinoma.

Topics: Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Division; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Liver Neoplasms; Mitogen-Activated Protein Kinases; Phosphorylation; Polyenes; Polyunsaturated Alkamides; Protein Prenylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; ras Proteins; Signal Transduction; Tumor Cells, Cultured

Recently, many inhibitors of farnesyl protein transferase (FPTase) have been identified. Some of them interrupt cell growth in addition to Ras and nuclear lamin processing of Ras-transformed cells. We have tested the effect of the FPTase inhibitors manumycin, an analogue of farnesyl diphosphate, and KT7595, a gliotoxin derivative, on Ras farnesylation, DNA synthesis and the anchorage-dependent and -independent growth of human colon carcinoma (LoVo), hepatoma (Mahlavu and PLC/PRF/5) and gastric carcinoma (KATO III). Both drugs severely inhibited DNA synthesis, cellular proliferation and Ras farnesylation in LoVo and moderately reduced them in Mahlavu and PLC/PRF/5 but not in KATO III. Complete sequencing of ras genes, however, revealed that LoVo and KATO III have activated Ki-ras and activated N-ras, respectively, whereas Mahlavu and PLC/PRF/5 have no activated ras. We next checked whether the inhibition of the cellular proliferation is due to the blocking of nuclear lamin function. Neither drug disturbed lamin farnesylation and localization, as demonstrated using metabolic labelling, immunoblotting and indirect immunofluorescence. These results indicate that manumycin and KT7595 can inhibit Ras farnesylation and cell growth without disturbing the farnesylation and localization of the lamins on human tumour cell lines.

Topics: Alkyl and Aryl Transferases; Carcinoma, Hepatocellular; Cell Division; Colonic Neoplasms; DNA, Neoplasm; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Fluorescent Antibody Technique, Indirect; Gliotoxin; Humans; Immunoblotting; Lamins; Liver Neoplasms; Neoplasms; Nuclear Proteins; Polyenes; Polyunsaturated Alkamides; Protein Prenylation; ras Proteins; Stomach Neoplasms; Tumor Cells, Cultured

So far, treatment with anti-cancer agents has failed to achieve satisfactory results in hepatocellular carcinoma. In the process of hepatocarcinogenesis, ras has been shown to play a role. ras requires a farnesyl moiety for activation. It has been found that UCFI-C (manumycin), an antibiotic, inhibits farnesyl protein transferase, an enzyme that catalyzes farnesylation. Therefore, we investigated the effects of UCFI-C on cell growth, prenylation of cellular proteins including ras and Rapl, MAP kinase activity, activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and synthesis of cholesterol in a ras-activated human hepatoma cell line, Hep G2. Treatment with varying concentrations of UCF1-C(10-30 microM for 24 and 72 hr resulted in a time- and dose-dependent inhibition of cell numbers. 3H-Thymidine incorporation was also inhibited in a dose-dependent manner, with 50% inhibition after 44 hr being observed at a concentration of 17 microM. UCFI-C dose-dependently inhibited ras farnesylation and MAP kinase activity, but did not decrease Rap 1++ geranylgeranylation or prenylation of 21-to 26-kDa proteins. Neither the activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase nor cholesterol synthesis were inhibited. These results suggest that UCFI-C antagonizes the growth of Hep G2 via the suppression of ras farnesylation and could be a lead for the development of new anti-cancer agents blocking the function of oncogenic ras associated with human cancer, including hepatocellular carcinoma.

Topics: Alkyl and Aryl Transferases; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Hepatocellular; Cell Division; Cholesterol; GTP-Binding Proteins; Humans; Hydroxymethylglutaryl CoA Reductases; Polyenes; Polyunsaturated Alkamides; rap GTP-Binding Proteins; Transferases; Tumor Cells, Cultured