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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.

Analogs of geranylgeranyl diphosphate (GGdP) have been demonstrated to inhibit the geranylgeranylation of proteins, producing cytotoxic activity in human prostate cancer cells. A detailed study is reported on the programmed cell death in vitro of human exocrine pancreas cancer cells (MIA PaCa-2) induced by the most active compound of this series of geranylgeranylation inhibitors, the dipotassium salt of (E,E,E)[2-oxo-2-[[(3,7,11,15-tetramethyl-2, 6,10,14-hexadecatetraenyl)-oxy]amino]ethyl] phosphonic acid (BAL 9504), using transmission and scanning electron microscopy (SEM). The results show that, after 72 h of treatment with BAL 9504, 25 microM, most MIA PaCa-2 cells display the typical morphological features of apoptosis, including condensation of nuclear chromatin, dilation of endoplasmic reticulum, and fragmentation of both nucleus and cytoplasm, giving rise to small membrane-bound vesicles (apoptotic bodies); surface protrusions and blebs are well demonstrated by SEM. The electrophoresis showed the presence of various bands corresponding to fragmented DNA of 180 base pairs, or multiples of this length, thus indicating that BAL 9504 effectively induces apoptosis. The present study provides the first evidence that inhibition of protein geranylgeranylation produces apoptosis in human MIA PaCa-2 exocrine pancreas cancer cells.

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 p21ras farnesylation with a 50% inhibitory concentration (IC[SUB50]) 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[SUB50])=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[SUB50] = 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 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. [ABSTRACT FROM AUTHOR]