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 (IC 50 ) of 2.51 ± 0.11 μ M and 2.68 ± 0.20 μ M , respectively, while the geranylgeranylation of p21 rhoA and p21 rap1 was not affected. Manumycin dose-dependently inhibited (IC 50 = 2.40 ± 0.67 μ M ) the phosphorylation of the mitogen-activated protein kinase/extracellular-regulated kinase 2 (p42MAPK/ERK2), the main cytoplasmic effector of p21 ras , as well as COLO320-DM cell growth (IC 50 = 3.58 ± 0.27 μ M ) without affecting the biosynthesis of cholesterol. Mevalonic acid (MVA, 100 μ M ), a substrate of the isoprenoid synthesis, was unable to protect COLO320-DM cells from manumycin cytotoxicity. Finally, manumycin 1–25 μ M 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 p21 ras 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. © 2000 Cancer Research Campaign

The present study investigates the effect of the somatostatin analogue octreotide acetate (SMS 201-995) on experimental angiogenesis in vitro and in vivo. Octreotide reduced the proliferation of human HUV-EC-C endothelial cells (mean, -45.8% ver sus controls at 10(-9) M; P <0.05) as well as the density of the vascular network of the chick chorioallantoic membrane (mean, -35.7% versus controls at 50 mu g; P <0.05). Furthermore, octreotide significantly inhibited chick chorioallantoic membrane neovascularization by the human MCF-10A(int-2) mammary cells secreting the angiogenic protein FGF-3. The proliferation of endothelial and smooth muscle cells from rat aorta explants on fibronectin was reduced by octreotide 10(-8) M (mean, -32.6% versus controls; P <0.05), and a similar effect was produced on cells sprouting from explants cultured in fibrin (mean, -52.9% versus controls; P <0.05). Topical administration of octreotide 10 mu g/day for 6 days inhibited rat cornea neovascularization induced by AgNO3/KNO3 (mean, -50.6% versus controls; P <0.05). Octreotide 40 mu g/day i.p was tested on angiogenesis in rat mesentery obtained by i.p. injections of compound 48/80, a mast cell degranulating agent, or conditioned medium from MCF-10A(int-2) cells and was able to reduce the extent of neovascularization (mean, -45.6 and -64.1%, respectively, versus controls; P <0.05). These data provide evidence that octreotide is an inhibitor of experimental angiogenesis in vitro and in vivo.

The objective is to evaluate whether an ex-vivo model can be used to test intracellular contrast agents for MR imaging of the liver. T1 weighted inversion recovery, proton density spin echo and T2* weighted gradient echo images of the liver were acquired at 0.5 T in 10 rats before and 30 min after intravenous injection of 0.075 mmol/kg Gadolinium benzyloxypropionictetraacetate (Gd-BOPTA, n = 5) or 0.015 mmol/kg dextran magnetite (DM, n = 5), Four additional animals served as controls. After exsanguination and perfusion with saline and formalin, specimens of the liver and brain were embedded in an agar gel and examined with MR imaging one to three weeks later using the same protocol. In-vivo, the mean liver signal enhancement caused by Gd-BOPTA in T1, proton density and T2* weighted images was +23%, +28% and -70%, respectively. The mean liver signal enhancement caused by DM was -71%, -76% and -94%. In-vitro, no signal change was seen in the brain of animals injected with Gd-BOPTA and DM as compared to controls. Liver signal was increased by Gd-BOPTA and decreased by DM. Mean liver enhancement rate induced by Gd-BOPTA was +22%, +5% and +27% for T1, proton density and T2* weighted images, respectively. Mean liver enhancement rate induced by DM was -27%, -19% and -31%. MR imaging signal changes induced by liver intracellular contrast agents are still appreciable in an ex-vivo model. The latter might be useful for for preliminary investigation of intracellular contrast agents for MR imaging of the liver.

In the present study, the ability of suramin 18 mg/kg i.p. twice a week to induce chronic neurotoxicity in rats was investigated. After 20 weeks of suramin treatment, morphological analysis of nerve fibers demonstrated that 57.7±3.2% of them presented vesicular disruption of myelin sheaths; their thickness was 0.23±0.07 μm in suramin-treated rats with respect to 0.43±0.07 μm of controls (P<0.05). To investigate the interaction between suramin and nerve tissue, the binding of the drug to partially purified myelin P0 protein obtained from sciatic nerves was analysed. The percentage of suramin bound to rat myelin P0 protein was 94.0±9.5%; this value was decreased to 55.0±7.6% when heparan sulfate was added to the myelin protein suspension before suramin. The analysis of tissue drug concentrations at 5, 10 and 20 weeks of treatment showed that suramin accumulated into the sciatic nerve in a time-dependent fashion (130.8±18.1, 219.7±17.1 and 449.3±15.6 μg/g of tissue, respectively). In conclusion, suramin induces a chronic peripheral neurotoxicity in rats characterized by myelin damage and high tissue levels of the drug. The high affinity of suramin for partially purified myelin P0 protein suggests a possible mechanism for drug-induced toxicity.

This chapter discusses substrate interactions of protein prenyltransferases. The posttranslational modification of proteins by isoprenoids facilitates protein–membrane and protein–protein interactions. This modification is common in a variety of proteins. It is estimated that as much as 0.5% of mammalian brain proteins are prenylated. Among the known prenylated proteins are most of the small guanosine triphosphate (GTP)-binding proteins, the γ subunits of trimeric G proteins, and nuclear lamins. Protein prenylation is likely to affect a wide variety of cellular functions. Prenylation of proteins is catalyzed by protein prenyltransferases. In the reaction a thioether bond is formed between the sulfhydryl group of a cysteine residue, located near the carboxy terminus of the acceptor protein, and either a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprenoid. The nature of the prenyl modification is highly dependent on the specificity of the prenyltransferases. The three protein prenyltransferases isolated are (1) farnesyltransferase (FTase), (2) geranylgeranyltransferase I (GGTase I), and (3) geranylgeranyltransferase II (GGTase II).The determination of farnesyl pyrophosphate binding assay using nonisolated enzyme-[3H] farnesyl pyrophosphate complex is described in the chapter.

The refractoriness of prostate cancer to androgen suppression is the landmark of clinically aggressive disease. In this study, the androgen-dependent LNCaP prostate cancer cells were transfected with the mutated c-Ha-ras gene from the T24 human bladder cancer. The derivative clone overexpressing T24-ras (LNCaP(T24-ras)) proliferated in androgen-depleted medium and showed increased growth. Protein isoprenylation and p21ras farnesylation in LNCaP(T24-ras) cells were tested in the presence of phenylacetate to document a possible relationship with the drug-induced inhibition of cell proliferation. Phenylacetate is a differentiation inducer that down-regulates in vitro the expression of the myc oncogene and activates the human peroxisome proliferator-activated nuclear receptor involved in cell growth regulation. The drug inhibited protein isoprenylation and p21ras farnesylation in LNCaP(T24-ras) cells; IC50 values were 3.1 and 3.3 mM, respectively, compared with controls. The drug reduced the cellular levels of endogenous farnesyl-PP (mean IC50 = 3.5 mM) and inhibited activation of the p21ras downstream target, p42(MAPK)/ERK2. LNCaP(T24-ras) was more sensitive than the parental line to both growth inhibition (mean IC50 = 3.01 and 7.1 mM, respectively) and apoptosis by phenylacetate. Exogenous farnesyl- and geranylgeranyl-PP indeed reduced the effects of the drug on proliferation and apoptosis in LNCaP(T24-ras) cells. In conclusion, the inhibition of protein isoprenylation and p21ras farnesylation by phenylacetate resulted in increased chemosensitivity of the androgen-independent LNCaP(T24-ras) cells compared with LNCaP, and this effect might contribute to the pharmacological activity of the drug.

The pharmacokinetics of single intraperitoneal doses of paclitaxel (18 and 36 mg/kg) in mice were investigated in the present study. The analysis of drug concentrations by HPLC indicated that the plasma Cmax (13.0 +/- 3.1 and 25.7 +/- 2.8 micrograms/ml, respectively) were reached at the 2nd hr. The values of CL were low (0.06 and 0.1 ml/min, respectively), and t1/2 beta values of 3.0 and 3.7 hr were found, after 18 and 36 mg/kg, respectively. The highest tissue concentrations were observed in the liver (50.2 +/- 3.1 and 92.0 +/- 9.5 micrograms/g respectively), followed by the pancreas (39.3 +/- 9.9 micrograms/g) and the ovary (53.4 +/- 5.6 micrograms/g) after 18 and 36 mg/kg, respectively. In the case of the colic tissue, paclitaxel Cmax were 14.4 +/- 0.8 and 32.8 +/- 3.5 micrograms/g at the 3rd hr, respectively, with sustained drug levels still detectable 24 hr after treatment. Paclitaxel Cmax values of 12.7 +/- 3.0 and 53.4 +/- 5.6 micrograms/g were detected in the ovary after 18 and 36 mg/kg, respectively. The overall results provide evidence that, after intraperitoneal administration, paclitaxel concentrates in peritoneal organs; however, the intraperitoneal route does not prevent systemic drug exposure, allowing high and sustained levels of paclitaxel also in several extraperitoneal tissues.

The activity of the synthetic somatostatin analogue SMS 201-995 was investigated in vitro on the growth of SW480 and SW620 human colon adenocarcinoma cell lines. The inhibition of cell proliferation was significant in SW480 cells (-19.6+/-1.4% at SMS 201-995 10(-9) M, P<0.05), but not in SW620 cells (-5.5+/-0.8% at SMS 201-995 10(-8) M) as compared to untreated cultures, Moreover, SMS 201-995 10(-8) M decreased the mitogenic effect of epidermal growth factor (EGF) on the SW480 cell line (-26.6+/-3.4% vs. cells exposed to EGF 10 ng ml(-1) alone, P<0.05). The effect of combining SMS 201-995 plus the cytokines interleukin-2 (IL-2) or gamma-interferon (gamma-IFN) on SW480 and SW620 cancer cell growth was also evaluated. The treatment produced a synergistic antiproliferative effect against SW620 cells as compared to untreated cultures, with growth inhibition being -20.2+/-1.2 and -19.3+/-1.3%, at SMS 201-995 10(-8) M plus IL-2 or gamma-IFN 100 IU ml(-1), respectively, but did not increase the activity of SMS 201-995 against the SW480 cells. In conclusion, the effect of SMS 201-995 on colon cancer cell growth can be enhanced by its combination with cytokines in SW620 but not in SW480 colon adenocarcinoma cells. (C) 1995 The Italian Pharmacological Society

The purpose of this study was to examine the cytotoxicity and cardiotoxicity of new doxorubicin (DXR) derivatives, 3'-deamino-3'-(2-methoxy-4- morpholinyl)DXR (MRA-MT), and 4'-deoxy-4'-iodo-doxorubicin (IDXR), comparing them to doxorubicin (DXR). Both anthracycline derivatives were approximately 1.5- to 9-fold more active than DXR in inhibiting the colony-formation ability of DU145, HOS, and A2780 human cancer cell lines. Anesthetized rats given a single intravenous (i.v.) dose of DXR 10 mg/kg showed significant changes in both ECG (S alpha T segment and QRS complex widening) and hemodynamic parameters (impairment in systemic arterial dP/dtmax systolic and diastolic blood pressure), whereas animals that received MRA-MT (0.1 and 0.3 mg/kg) had no significant signs of acute cardiotoxicity. In this case the animals treated with IDXR 1.2 mg/kg showed alterations in the ECG as the animals treated with DXR. In the chronic cardiotoxicity study, some animals received MRA-MT (0.03 mg/kg i.v. once a week for 3 weeks) and others IDXR (4 mg/kg once a week for 3 weeks). They did not show any alteration in ECG and cardiac histological picture. By contrast, DXR (3 mg/kg i.v. once a week for 3 weeks) induced a severe cardiomyopathy, characterized by progressive widening of S alpha T segment, increase in T wave, and histological damage consisting of vacuolations and loss of myofibrils. These results suggest that MRA-MT and IDXR are more active in vitro and markedly less cardiotoxic in vivo than DXR.

the last years, several studies have demonstrated that ras genes are involved in the signal transduction from transmem-brane receptors to intracellular effectors, thus controlling cell differentiation and growth (Marshall, 1996). Alteration of the normal biologic function of ras proteins might be associated with the acquisition of a neoplastic phenotype of cells (Lowy and Willumsen, 1993). Mutational activation of ras oncogenes is present in up to 90% of pancreatic cancers and 50% of all cases of colorectal cancer (Bos, 1989), two neoplasms poorly responsive to the treatment with traditional chemotherapeutic agents. Moreover, the mutational activation of ras is associated with a poor prognosis and a shorter survival of patients (Rodenhuis et al, 1997). Thus, the inhibition of ras protein function could be an effective pharmacological approach in cancer chemotherapy. Proteins belonging to the ras superfamily exert their functions when they are localized to specific cellular compartments, and this targeting is made possible by post-translational modifications (Marshall, 1993). The first of them consists of the farnesylation of the cysteine residue at the carboxyterminal tetrapeptide CAAX, where C is cysteine, A any aliphatic amino acid, and X methionine or serine (Maltese, 1990). This step is crucial, because its inhibition causes the interruption of the subsequent processing of the cytosolic immature ras proteins, thus impairing their biological functions (Gibbs, 1994). In this regard, numerous attempts have been directed to the identification of chemotherapeutic agents Among them, manumycin is a promising compound derived from Streptomyces parvulus and characterized by Hara et al (1993). Manumycin acts via the specific inhibition of the farnesyl:protein transferase (FPTase), as demonstrated by the reversion of a ras-dependent phenotype in the worm Caenorhabditis elegans (Hara and Han, 1995) and exerts antiproliferative effect on human tumour cells which harbour a mutated K-ras gene (Nagase et al, 1996). However, the issue of therapeutic targeting of normal ras proteins has not been addressed and data are lacking concerning the ability of FPTase inhibitors to suppress the proliferation of tumour cells with wild-type K-ras genes. In the present work, the effect of manumycin was evaluated on the processing of isoprenylated proteins of the ras superfamily, ras signal transduction pathway and cell death in the cell line COLO320-DM derived from a human colorectal carcinoma. METHODS Drugs and chemicals Antipain, leupeptin, aprotinin, sodium dodecyl sulphate (SDS) and proteinase K were purchased from Boehringer Mannheim GmbH (Mannheim, Germany). RPMI-1640 and fetal bovine serum (FBS) were from HyClone (Cramlington, …