Manumycin inhibits ras signal transduction pathway and induces apoptosis in COLO320-DM human colon tumourcells

Abstract

SummaryThe 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 p21ras farnesylation with a 50% inhibitory concentration (IC 50 ) of 2.51 ± 0.11 µM and 2.68 ± 0.20 µM, respectively, while the geranylgeranylation of p21rhoA and p21rap1 was not affected. Manumycin dose-dependently inhibited (IC 50 = 2.40 ± 0.67 µM) 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 (IC 50 = 3.58 ± 0.27 µM) without affecting the biosynthesis of cholesterol. Mevalonic acid (MVA, 100 µM), a substrate of the isoprenoid synthesis, was unable to protect COLO320-DM cells from manumycin cytotoxicity. Finally, manumycin 1-25 µM for 24-72 h induced oligonucleosomal fragmentation in a dose-and timedependent 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.

Inhibition of protein farnesylation enhances the chemotherapeutic efficacy of the novel geranylgeranyltransferase inhibitor BAL9611 in human colon cancer cells

“…The inhibitory effect of manumycin on p21ras farnesylation and signal transduction through p42ERK2/MAPK pathway has been previously documented in cell lines harbouring wild-type (Di Paolo et al, 2000) or mutated K-ras gene (Kainuma et al, 1997). The present evidence that the phosphorylation of p42ERK2/MAPK may be decreased by the GGTase inhibitor BAL9611 suggests that p42ERK2/MAPK activity may be modulated by geranylgeranylated proteins, such as p21rhoA.…”

“…Film densities of protein blots were examined with the Kontron Imaging analysis system KS300 (Kontron Elektronic, Eching, Germany) connected to a TK-1280E colour video camera (JVC, Tokyo, Japan), and expressed as mean percentage ± standard error (SE) of arbitrary units of grey values with respect to controls (Di Paolo et al, 2000). In order to compensate for the decrease in protein synthesis as a consequence of treatment of cells with test compounds, the inhibitory effect of BAL9611 and manumycin on p21ras and p21rhoA isoprenylation as well as on p42ERK2/MAPK phosphorylation was expressed as the ratio of isoprenylated/phosphorylated versus non-isoprenylated/non-phosphorylated bands.…”

Fluvastatin synergistically enhances the antiproliferative effect of gemcitabine in human pancreatic cancer MIAPaCa-2 cells

“…MIAPaCa-2 cells were maintained in DMEM medium, supplemented with 10% FBS, 2.5% HS, penicillin (50 IU ml À1 ), streptomycin (50 mg ml À1 ) and L-glutamine (2 mM). The human colon cancer cell line COLO320-DM, a cell line with wild-type K-ras (Di Paolo et al, 2000), was from ATCC and maintained in RPMI 1640 medium supplemented with 10% FBS, penicillin and L-glutamine. Cells were routinely grown in 75 cm 2 tissue culture flasks and kept in a humidified atmosphere of 5% CO 2 at 371C.…”

“…Film densities of protein immunoblots and apoptosis assays were quantified through video imaging densitometry with the KS300 version 1.2. software (Kontron Elektronic, Eching, Germany), and expressed as arbitrary units of mean gray values (optical density), in the range of 0 -255, where 0 was black and 255 was white (Di Paolo et al, 2000). Results were expressed as the mean7s.e.…”

Manumycin A induces apoptosis in malignant pleural mesothelioma through regulation of Sp1 and activation of the mitochondria-related apoptotic pathway

“…Recently, the anti-neoplastic activity of Manu A has been demonstrated in various experimental systems (18). It exerts antitumor activity against a variety of cancer cells such as human pancreatic cancer cells (19), anaplastic thyroid cancer cells (20,21), human colon tumor cells (22) and human hepatocellular carcinoma HepG2 cells (23), medulloblastoma cells (24,25), leukemic cells (26), lymphoid tumor and myeloma cell lines (27,28). However, little is known concerning the anticancer mechanisms of Manu A by which it can induce apoptosis in MPM cells.…”