manumycin and Colonic-Neoplasms

Salternamides A-D (1-4), the first secondary metabolites discovered from saltern-derived actinomycetes, were isolated from a halophilic Streptomyces strain isolated from a saltern on Shinui Island in the Republic of Korea. The planar structures of the salternamides, which are new members of the manumycin family, were elucidated by a combination of spectroscopic analyses. The absolute configurations of the salternamides were determined by chemical and spectroscopic methods, including the modified Mosher's method, J-based configuration analysis, and circular dichroism spectroscopy. Salternamide A (1), which is the first chlorinated compound in the manumycin family, exhibited potent cytotoxicity against a human colon cancer cell line (HCT116) and a gastric cancer cell line (SNU638) with submicromolar IC50 values. Salternamides A and D were also determined to be weak Na(+)/K(+) ATPase inhibitors.

Topics: Actinobacteria; Antineoplastic Agents; Circular Dichroism; Colonic Neoplasms; Drug Screening Assays, Antitumor; Humans; Inhibitory Concentration 50; Molecular Structure; Polyenes; Polyunsaturated Alkamides; Republic of Korea; Salt-Tolerant Plants; Sodium-Potassium-Exchanging ATPase; Streptomyces

Our previous studies demonstrated that manumycin (a farnesyltransferase inhibitor) enhanced the antineoplastic activity and induction of apoptosis when combined with paclitaxel against anaplastic thyroid cancer cells. We found that manumycin induces endogenous expression of p21 Waf-1 in anaplastic thyroid cancer cells. Manumycin increased the activity of the p21promoter, the level of p21mRNA, and the amount of p21 protein. We hypothesized that p21 had a proapoptotic effect in cells treated with manumycin, or paclitaxel, or both agents. By measuring viability and caspase-3 activity, we found that stably transfected KAT-4 cells with p21 cDNA under the control of a metallothionein promoter were more sensitive to manumycin alone, paclitaxel alone, and manumycin plus paclitaxel when p21was induced. The increased sensitivity of the cells with induced p21 was associated with an increase in caspase-3 activity (i.e. apoptosis). We also found that cells with both p21 alleles deleted were less sensitive to manumycin plus paclitaxel than its wild-type parent cells. Expression of p21 per se did not induce apoptosis but enhanced the cytotoxic effects of manumycin and paclitaxel. These findings suggested that p21 might be required to maintain cell sensitivity to the cytotoxic effects of manumycin and paclitaxel.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Caspases; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Gene Expression; Humans; Mutagenesis; Oligonucleotide Array Sequence Analysis; Paclitaxel; Polyenes; Polyunsaturated Alkamides; Thyroid Neoplasms; Transfection; Tumor Cells, Cultured; Zinc

Proteins belonging to the ras superfamily are involved in cell proliferation of normal and neoplastic tissues. To be biologically active, they require post-translational isoprenylation by farnesyl-transferase and geranylgeranyl-transferase. Enzyme inhibition by drugs may thus represent a promising approach to the treatment of cancer. Therefore, the combined effect of BAL9611, a novel inhibitor of geranylgeranylation, and manumycin, a farnesyl-transferase inhibitor, was evaluated on the SW620 human colon cancer cell line which harbours a mutated K-ras gene. BAL9611 and manumycin dose-dependently inhibited SW620 cell growth with 50% inhibitory concentration (IC(50)) of 0.47 +/- 0.03 and 5.24 +/- 1.41 microM (mean +/- SE), respectively. The isobologram analysis performed at the IC(50)level revealed that the combined treatment was highly synergistic with respect to cell growth inhibition. BAL9611 and manumycin were able to inhibit the geranylgeranylation of p21rhoA and farnesylation of p21ras; both drugs inhibited p42ERK2/MAPK phosphorylation, but their combination was more effective than either drug alone. Moreover, the enhanced inhibition of cell growth in vitro by the BAL9611-manumycin combination was also observed in vivo in CD nu/nu female mice xenografted with SW620 tumours. Finally, both drugs were able to induce cell death by apoptosis in vitro and in vivo, as demonstrated by perinuclear chromatin condensation, cytoplasm budding and nuclear fragmentation, and interoligonucleosomal DNA digestion. In conclusion, the inhibition of protein farnesylation enhances the chemotherapeutic effect of BAL9611 in vitro and in vivo in a synergistic fashion, as a result of the impairment of post-translational isoprenylation of proteins and phosphorylation of p42ERK2/MAPK, whose activation is associated with post-translational geranylgeranylation and farnesylation of p21rhoA and p21ras.

Topics: Alkyl and Aryl Transferases; Animals; Cell Division; Cell Transformation, Neoplastic; Colonic Neoplasms; Enzyme Inhibitors; Female; Humans; Mice; Organophosphonates; Polyenes; Polyunsaturated Alkamides; Protein Prenylation; Transplantation, Heterologous; Tumor Cells, Cultured

The aim of the present study was to assess the cytotoxicity of manumycin, a specific inhibitor of farnesyl:protein transferase, as well as its effects on protein isoprenylation and kinase-dependent signal transduction in COLO320-DM human colon adenocarcinoma which harbours a wild-type K-ras gene. Immunoblot analysis of isolated cell membranes and total cellular lysates of COLO320-DM cells demonstrated that manumycin dose-dependently reduced p21 ras farnesylation with a 50% inhibitory concentration (IC50) of 2.51 +/- 0.11 microM and 2.68 +/- 0.20 microM, respectively, while the geranylgeranylation of p21 rhoA and p21rap1 was not affected. Manumycin dose-dependently inhibited (IC50 = 2.40 +/- 0.67 microM) the phosphorylation of the mitogen-activated protein kinase/extracellular-regulated kinase 2 (p42MAPK/ERK2), the main cytoplasmic effector of p21ras, as well as COLO320-DM cell growth (IC50 = 3.58 +/- 0.27 microM) without affecting the biosynthesis of cholesterol. Mevalonic acid (MVA, 100 microM), a substrate of the isoprenoid synthesis, was unable to protect COLO320-DM cells from manumycin cytotoxicity. Finally, manumycin 1-25 microM for 24-72 h induced oligonucleosomal fragmentation in a dose- and time-dependent manner and MVA did not protect COLO320-DM cells from undergoing DNA cleavage. The present findings indicate that the inhibition of p21ras processing and signal transduction by manumycin is associated with marked inhibition of cell proliferation and apoptosis in colon cancer cells and the effect on cell growth does not require the presence of a mutated ras gene for maximal expression of chemotherapeutic activity.

Topics: Alkyl and Aryl Transferases; Apoptosis; Base Sequence; Cholesterol; Colonic Neoplasms; DNA Primers; Genes, ras; Humans; Mitogen-Activated Protein Kinases; Mutation; Phosphorylation; Polyenes; Polyunsaturated Alkamides; 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